Blood factor monitoring assay and uses thereof

ABSTRACT

The present disclosure provides methods and compositions for diagnosing and treating subject having a bleeding disorder. The disclosed methods comprise contacting a sample, e.g., a blood or plasma sample obtained from the patient, with an activation mixture comprising an activated coagulation factor and a phospholipid mixture, wherein the activation mixture is dried onto a solid substrate. Also provided is a global hemostasis test based on the integration of clotting time (Ct) and pharmacokinetics data. The methods and compositions presented can be applied to point-of-care diagnostic systems.

REFERENCE TO A SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The content of the electronically submitted sequence listing (Name:2159_3840001_SequenceListing.txt; Size: 112,914 bytes; and Date ofCreation: Jan. 16, 2015) is herein incorporated by reference in itsentirety.

BACKGROUND

Field of the Invention

The present invention relates generally to the field of therapeutics forhemostatic disorders.

Background Art

Hemophilia is a bleeding disorder in which blood clotting is disturbedby a lack of certain plasma clotting factors in the coagulation cascade(FIG. 1). Hemophilia A and Hemophilia B are two different types ofhemophilia that are caused by deficiencies in Factor VIII (FVIII) andFactor IX, respectively.

Hemophilia A is characterized by spontaneous hemorrhage and excessivebleeding after trauma. Over time, the repeated bleeding into muscles andjoints, which often begins in early childhood, results in hemophilicarthropathy and irreversible joint damage. This damage is progressiveand can lead to severely limited mobility of joints, muscle atrophy andchronic pain (Rodriguez-Merchan, E. C., Semin. Thromb. Hemost. 29:87-96(2003), which is herein incorporated by reference in its entirety).

Hemophilia B (also known as Christmas disease) is one of the most commoninherited bleeding disorders in the world. It results in decreased invivo and in vitro blood clotting activity and requires extensive medicalmonitoring, throughout the life of the affected individual. In theabsence of intervention, the afflicted individual will suffer fromspontaneous bleeding in the joints, which produces severe pain anddebilitating immobility; bleeding into muscles results in theaccumulation of blood in those tissues; spontaneous bleeding in thethroat and neck can cause asphyxiation if not immediately treated; renalbleeding; and severe bleeding following surgery, minor accidentalinjuries, or dental extractions also are prevalent.

Treatment of hemophilia is by replacement therapy targeting restorationof Factor VIII and Factor IX activity. Treatment of hemophilia A is byreplacement therapy targeting restoration of FVIII activity to 1 to 5%of normal levels to prevent spontaneous bleeding (Mannucci, P. M., etal., N. Engl. J. Med. 344:1773-1779 (2001), which is herein incorporatedby reference in its entirety). There are plasma-derived and recombinantFVIII products available to treat bleeding episodes on-demand or toprevent bleeding episodes from occurring by treating prophylactically.Based on the half-life of these products treatment regimens requirefrequent intravenous administration. Such frequent administration ispainful and inconvenient.

Treatment of hemophilia B occurs by replacement of the missing clottingfactor by exogenous factor concentrates highly enriched in Factor IX,but is also problematic. Generating such a concentrate from blood isfraught with technical difficulties. Purification of Factor IX fromplasma (plasma derived Factor IX; pdFIX) almost exclusively yieldsactive Factor IX. However, such purification of factor IX from plasma isvery difficult because Factor IX is only present in low concentration inplasma (5 ug/mL. Andersson, Thrombosis Research 7: 451 459 (1975).Further, purification from blood requires the removal or inactivation ofinfectious agents such as HIV and HCV. In addition, pdFIX has a shorthalf-life and therefore requires frequent dosing. Recombinant factor IX(rFIX) is also available, but suffers from the same short half-life andneed for frequent dosing (e.g., 2-3 times per week for prophylaxis) aspdFIX. rFIX also has a lower incremental recovery (K value) compared topdFIX, which necessitates the use of higher doses of rFIX than those forpdFIX.

Reduced mortality, prevention of joint damage and improved quality oflife have been important achievements due to the development ofplasma-derived and recombinant Factor VIII and Factor IX products.Prolonged protection from bleeding would represent another keyadvancement in the treatment of hemophilia patients. In order to addressthis need, recombinant Factor VIII and Factor IX proteins expressed asFc fusions are in development. However, methods of determiningappropriate dosage of these products, which have unique pharmacokineticproperties in humans have not yet been developed. Therefore, thereremains a need for improved methods of treating hemophilia due to FactorVIII and Factor IX deficiencies that are more tolerable and moreeffective than current therapies.

Coagulation assays have gained acceptance as an important tool formanagement of patients being treated for coagulation disorders. Thesetreatments are also applicable to patients on anticoagulation therapyfor the prevention of clots in their blood vessels. In these assays, asample of the patient's blood or plasma is tested for coagulation timeor “clotting time” which time is related to the amount of coagulationfactors in the patient's blood (or to the patient's dosage ofanticoagulant in the case of patients undergoing antocoagulationtherapy). Coagulation assays are also required prior to surgicalprocedures even for patients not suffering from bleeding disorders or onanticoagulation therapy. This is because the medical professionals needto clearly know the bleeding susceptibility before they are operated on.

A variety of coagulation test are presently in use and among the mostpopular is the “Activated Partial Thromboplastin Time” (aPTT) test (seeFIG. 2). Blood coagulation tests have tended to be complex, and the bulkof them are performed generally in centralized clinical laboratories.Clinical or a doctor's office visits or a regular basis to monitorcoagulation factor levels can be very inconvenient and expensive. Mostof apparatus and methods known for measuring coagulation time in bloodsamples cannot be used for home testing (see, e.g., U.S. Pat. Nos.3,695,842; 3,836,333; 4,197,734; 3,486,859; 4,797,369; 3,890,098;4,725,554; 5,284,624; 3,951,606; 4,659,550; and 5,302,348). Thedisadvantages of these methods, beside cost and the challenge ofoperation, include the fact that most do not measure coagulationdirectly. The large blood volume requirements of some of these methodsmade them impractical for home use. Many of these methods are alsolimited by what kinds of coagulation tests they can perform due to thereagent chemistry requirements and the detectable signal generated.

BRIEF SUMMARY

The present disclosure provides a composition for the measurement ofcoagulation factor activity in a sample comprising an activatedcoagulation factor and a phospholipid mixture, wherein the compositionis dried onto a solid substrate. The present disclose also provides acomposition for the measurement of coagulation time in a samplecomprising an activated coagulation factor and a phospholipid mixture,wherein the composition is dried onto a solid substrate. In someaspects, the solid substrate is selected from the group consisting ofpaper, plastic, glass, ceramic material, metal, and combinationsthereof. In other aspects, the solid substrate is a surface on a teststrip, test stick, reaction chamber, cartridge, chip, well plate, orarray used in an apparatus to measure coagulation factor activity orcoagulation time.

In some aspects, the coagulation factor is selected from the groupconsisting of FVII, FVIII, and FIX. In other aspects, the coagulationfactor is a Factor VIII protein or a fragment, variant, or derivativethereof. In some aspects, the coagulation factor is a Factor IX proteinor a fragment, variant, or derivative thereof. In other aspects, theactivated coagulation factor is a Factor IXa protein or a fragment,variant, or derivative thereof. In some aspects, Factor IXa is presentin the composition prior to drying within a range of 0.01 to 0.05 U/mL.In other aspects, the activated coagulation factor is a Factor XIaprotein or a fragment, variant, or derivative thereof. In some aspects,Factor XIa is present in the composition prior to drying within a rangeof 0.01 to 0.05 U/mL.

In some aspects, the phospholipid mixture comprises 2 phospholipids. Inother aspects, the phospholipid mixture comprises 3 phospholipids. Insome aspects, the phospholipids are selected from the group consistingof phosphatidylcholine, phosphatidylserine, phosphatidylglycerol, andcombinations thereof. In other aspects, the phospholipids are naturalphospholipids, synthetic phospholipids, or combinations thereof. In someaspects, the phospholipid mixture comprises 70 mole-% ofphosphatidylcholine and 30 mole-% of phosphatidylserine. In otheraspects, the phospholipid mixture comprises 80 mole-% ofphosphatidylcholine, 10 mole-% of phosphatidylserine, and 10 mole-% ofphosphatidylglycerol. In some aspects, the phospholipid mixturecomprises 75 mole-% of phosphatidylcholine, 20 mole-% ofphosphatidylserine, and 5 mole-% of phosphatidylglycerol. In otheraspects, the phospholipid mixture further comprises cholesterol. In someaspects, the cholesterol content in the phospholipid mixture is fromabout 1 to about 20 mole-% of cholesterol.

In some aspects, the phospholipid mixture is in vesicle form. In otheraspects, the vesicles are small unilamellar vesicles. In some aspects,the composition further comprises divalent cations. In other aspects,the divalent cations are calcium ions. In some aspects, the sample isselected from the group consisting of whole blood, citrated orequivalently stabilized blood, plasma, or other fluid sample containingor suspected of containing a coagulation factor. In other cases, thesample is decalcified.

In some aspects, the measurement is carried in a point of care testsystem. In some aspects, the measurement is carried out in a mechanicalor optical analytical system.

The present disclosure provides a composition for the measurement of theFactor VIII activity of a Factor VIII protein or a fragment, variant, orderivative thereof in a sample comprising 80% of 0.1 mg/mL Factor IXaand 20% of a phospholipid mixture comprising 75 mole-% ofphosphatidylcholine, 20 mole-% of phosphatidylserine, and 5 mole-% ofphosphatidylglycerol, wherein said composition is dried onto a solidsubstrate. Also provided is a composition for the measurement of theFactor IX activity of a Factor IX protein or a fragment, variant, orderivative thereof in a sample comprising 80% Factor XIa suspension and20% of a phospholipid mixture comprising 75 mole-% ofphosphatidylcholine, 20 mole-% of phosphatidylserine, and 5 mole-% ofphosphatidylglycerol, wherein said composition is dried onto a solidsubstrate. The exact amount of FXIa needed varies depending on thespecific activity of this reagent and is titrated for optimal amount andcan include approximately 0.1 mg/mL.

The present disclosure also provides a kit for performing a measurementof coagulation factor activity or coagulation time in a samplecomprising a composition disclosed herein in one or more vials. Alsoprovided is a kit for performing a measurement of coagulation factoractivity or coagulation time in a sample comprising a compositiondisclosed herein in a non-dry form in one or more vials and instructionsfor drying said composition onto a solid substrate. The instantdisclosure also provides a sample holder for performing a bloodcoagulation assay, comprising a surface coated with any of theactivation mixtures disclosed herein. In some aspects, the sample holderis selected from the group consisting of a test strip, a test stick, areaction chamber, a cartridge, a chip, a well plate, and an array.

The present disclosure provides a method for determining clotting timein a patient having a bleeding disorder, comprising (a) contacting asample obtained from the patient with an activation mixture comprisingan activated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; and, (b) measuringthe time between the contacting of the activation mixture with the bloodsample and the onset of clotting, thereby calculating the clotting time(Ct).

Also provided is a method of treating a patient having a bleedingdisorder comprising (a) contacting a sample obtained from the patientwith an activation mixture comprising an activated coagulation factorand a phospholipid mixture, wherein the activation mixture is dried ontoa solid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct), wherein Ct indicates whether thepatient will benefit from administration of a treatment; and, (c)administering the treatment to the patient if Ct indicates that thepatient will benefit from administration of the treatment. The presentdisclosure also provides a method of treating a patient having ableeding disorder comprising (a) contacting a sample obtained from thepatient with an activation mixture comprising an activated coagulationfactor and a phospholipid mixture, wherein the activation mixture isdried onto a solid substrate; (b) measuring the time between thecontacting of the activation mixture with the sample and the onset ofclotting, thereby calculating the clotting time (Ct), wherein Ctindicates whether the patient will benefit from administration of atreatment; and, (c) instructing a healthcare provider to administer thetreatment to the patient if Ct indicates that the patient will benefitfrom administration of the treatment.

The present disclosure provides a method of optimizing a bleedingdisorder treatment in a patient comprising (a) contacting a sampleobtained from the patient with an activation mixture comprising anactivated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct),wherein Ct correlates with a therapeutically efficacious treatment; and,(c) administering an optimized treatment to the patient, wherein thetreatment is maintained or adjusted. Also provides is a method ofoptimizing a bleeding disorder treatment in a patient comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct), wherein Ct correlates with a therapeutically efficacioustreatment; and, (c) instructing a healthcare provider to optimize thetreatment administered, wherein the treatment is maintained or adjusted.

The present disclosure also provides a method of diagnosing whether apatient is in need of treatment for a bleeding disorder comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct), wherein Ct indicates whether the patient has a bleedingdisorder; and, (c) providing a treatment for the bleeding disorder ifthe patient is in need thereof. Also provided is a method of diagnosingwhether a patient is in need of treatment for a bleeding disordercomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct), wherein Ct indicates whether thepatient has a bleeding disorder; and, (c) instructing a healthcareprovider to provide treatment for the bleeding disorder if the patientis in need thereof.

The present disclosure also provides a method of monitoring the efficacyof a bleeding disorder treatment administered to a patient comprising(a) contacting a sample obtained from the patient with an activationmixture comprising an activated coagulation factor and a phospholipidmixture, wherein the activation mixture is dried onto a solid substrate;(b) measuring the time between the contacting of the activation mixturewith the sample and the onset of clotting, thereby calculating theclotting time (Ct); and, (c) comparing the measured Ct with the Ctobtained from a corresponding standard, wherein the standard isrepresentative of a therapeutically efficacious treatment, and wherein asimilarity between the patient's results and the standard is indicativeof efficacy of the patient's current treatment; and, (d) maintaining oradjusting the patient's treatment based on the relative differencebetween the patient's results and the corresponding standard. Alsoprovided is a method of monitoring the efficacy of a bleeding disordertreatment administered to a patient comprising (a) contacting a sampleobtained from the patient with an activation mixture comprising anactivated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating, the clotting time (Ct);(c) comparing the measured Ct with the Ct obtained from a corresponding,standard, wherein the standard is representative of a therapeuticallyefficacious treatment, and wherein a similarity between the patient'sresults and the standard is indicative of efficacy of the patient'scurrent treatment; and, (d) instructing a healthcare provider tomaintain or adjusting the patient's treatment based on the relativedifference between the patient's results and the corresponding standard.

The present disclosure also provides a method for determining acoagulation factor level in a bleeding disorder patient, comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate:

(b) measuring the time between the contacting of the activation mixturewith the sample and the onset of clotting, thereby calculating theclotting time (Ct), and, (c) correlating the Ct value with the level ofcoagulation factor in the sample. In some aspects, the correlationbetween Ct and coagulation factor level (% Factor) is calculatedaccording to the formula:Ct=A×Ln(% Factor)+B  [Formula I]wherein, for each coagulation factor, A is a constant valuecorresponding to the slope of a Ct versus coagulation factorconcentration dose-response, and B is patient-specific off-set value.

The present disclosure also provides a method for determining apharmacokinetic (PK) parameter in a bleeding disorder patient,comprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); and, (c) correlating a PK with thecalculated Ct value, thereby determining the value of the PK parameter.

The present disclosure provides a method of treating a patient having ableeding disorder comprising (a) contacting a sample obtained from thepatient with an activation mixture comprising an activated coagulationfactor and a phospholipid mixture, wherein the activation mixture isdried onto a solid substrate; (b) measuring the time between thecontacting of the activation mixture with the sample and the onset ofclotting, thereby calculating the clotting time (Ct); (c) determining aPK parameter based on Ct, wherein the PK parameter indicates that thepatient will benefit from administration of the treatment; and, (d)administering the treatment to the patient if the PK parameter indicatesthat the patient will benefit from administration of the treatment. Alsoprovides is a method of treating a patient having a bleeding disordercomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); (c) determining a PK parameter basedon Ct, wherein the PK parameter indicates that the patient will benefitfrom administration of the treatment; and, (d) instructing a healthcareprovider to administer the treatment to the patient if the PK parameterindicates that the patient will benefit from administration of thetreatment. Also provided is method of optimizing a bleeding disordertreatment in a patient comprising (a) contacting a sample obtained fromthe patient with an activation mixture comprising an activatedcoagulation factor and a phospholipid mixture, wherein the activationmixture is dried onto a solid substrate; (b) measuring the time betweenthe contacting of the activation mixture with the sample and the onsetof clotting, thereby calculating the clotting time (Ct); (c) determininga PK parameter based on Ct, wherein the PK parameter correlates with atherapeutically efficacious treatment; and, (d) administering anoptimized treatment to the patient, wherein the treatment is maintainedor adjusted. The present disclosure also provides a method of optimizinga bleeding disorder treatment in a patient comprising (a) contacting asample obtained from the patient with an activation mixture comprisingan activated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct);(c) determining a PK parameter based on Ct, wherein the PK parametercorrelates with a therapeutically efficacious treatment; and, (d)instructing a healthcare provider to administer an optimized treatmentto the patient, wherein the therapy is maintained or adjusted.

Also provided is a method of diagnosing whether a patient is in need oftreatment for a bleeding disorder comprising (a) contacting a sampleobtained from the patient with an activation mixture comprising anactivated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct);and, (c) determining a PK parameter based on Ct, wherein the PKparameter indicates whether the patient has a bleeding disorder; and,(d) providing treatment for the bleeding disorder if the patient is inneed thereof. Also provides is a method of diagnosing whether a patientis in need of treatment for a bleeding disorder comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct); and, (c) determining a PK parameter based on Ct, wherein thePK parameter indicates whether the patient has a bleeding disorder; and,(d) instructing a healthcare provider to provide therapy to treat thebleeding disorder if the patient is in need thereof.

The present disclosure also provides a method of monitoring the efficacyof a bleeding disorder treatment administered to a patient comprising(a) contacting a sample obtained from the patient with an activationmixture comprising an activated coagulation factor and a phospholipidmixture, wherein the activation mixture is dried onto a solid substrate;(b) measuring the time between the contacting of the activation mixturewith the sample and the onset of clotting, thereby calculating theclotting time (Ct); and, (c) determining a PK parameter based on Ct; (d)comparing the PK parameter with the PK obtained from a correspondingstandard, wherein the standard is representative of a therapeuticallyefficacious treatment, and wherein a similarity between the patient'sresults and the standard is indicative of efficacy of the patient'scurrent treatment; and, (e) maintaining or adjusting the patient'streatment based on the relative difference between the patient's resultsand the corresponding standard. Also provided is a method of monitoringthe efficacy of a bleeding disorder treatment administered to a patientcomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); (c) determining a PK parameter basedon Ct; (d) comparing the PK parameter with the PK obtained from acorresponding standard, wherein the standard is representative of atherapeutically efficacious treatment, and wherein a similarity betweenthe patient's results and the standard is indicative of efficacy of thepatient's current treatment; and, (e) instructing a healthcare providerto maintain or adjust the patient's treatment based on the relativedifference between the patient's results and the corresponding standard.

In some aspects, the PK is terminal half-life (HL). In other aspects,the PK is time to through (T). In some aspects, HL is calculatedaccording to the formula:HL=−0.693×(T ₂ −T ₁)×A/(Ct₁−Ct₂)  [Formula II]wherein, for each coagulation factor, A is a constant valuecorresponding to the slope of a Ct versus coagulation factorconcentration dose-response. T₁ and T₂ are times at which Ct ismeasured, and Ct₁ and Ct₂ are Ct values measured at T₁ and T₂,respectively.

In some aspects, T is calculated according to the formula:T=−1.44×HL/(A×(Ct_(measured)−Ct_(trough))  [Formula III]wherein for each coagulation factor A is a constant value correspondingto the slope of a Ct versus coagulation factor concentrationdose-response, and HL is the terminal half-life, Ct_(measured) is Ctmeasured at certain time point, and Ct_(trough) is patient-specific clottime at trough. In some aspects, the patient is administered a new doseof coagulation factor every T interval.

In some aspects, the sample is selected from the group consisting ofwhole blood, citrated or equivalently stabilized blood, plasma, or otherfluid sample containing or suspected of containing a coagulation factor.In some aspects, the sample is whole blood. In other aspects, the bloodis venous blood. In some aspects, the blood is fingerstick blood. Insome aspects, the sample is plasma. In some aspects, the sample isfrozen and thawed prior to contacting the sample with the activationmixture. In other aspects, the sample is has not been frozen and thawedprior to contacting the sample with the activation mixture. In someaspects, the sample is decalcified. In some aspects, the decalcifiedsample is recalcified prior to contacting the sample with the activationmixture. In other aspects, the decalcified sample is recalcified aftercontacting the sample with the activation mixture.

In some aspects, the sample further comprises an added purifiedcoagulation factor. In other aspects, the sample further comprises anadded inhibitor. In some aspects, the purified coagulation factor isselected from the group consisting of Factors II, Factor VII, FactorVIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII,Fibrinogen, vWF, Tissue Factor, and combinations thereof. In someaspects, the inhibitor is selected from the group consisting of CTI,aprotinin, ε-aminocaproic acid (EACA),D-Phenylalanyl-1-prolyl-1-arginine chloromethyl ketone-Factor VIIa(FPRCK-FVIIa), anti-coagulation factor monoclonal antibodies, andcombinations thereof. In some aspects, the sample is diluted withsubstrate sample. In specific aspects, one part of sample is dilutedwith three parts of substrate sample.

In some aspects, the activated coagulation factor is a Factor IXaprotein or a fragment, variant, or derivative thereof. In some aspects,Factor IXa is present in the composition prior to drying within a rangeof 0.01 to 0.05 U/mL. In other aspects, the activated coagulation factoris a Factor XIa protein or a fragment, variant, or derivative thereof.In some aspects, Factor XIa is present in the composition prior todrying within a range of 0.01 to 0.05 U/mL. In some aspects, thephospholipid mixture comprises 2 phospholipids. In some aspects, thephospholipid mixture comprises 3 phospholipids. In other aspects, thephospholipids in the phospholipid mixture are selected from the groupconsisting of phosphatidylcholine, phosphatidylserine,phosphatidylglycerol, and combinations thereof. In some aspects, thephospholipids are natural phospholipids, synthetic phospholipids, orcombinations thereof. In some aspects, the phospholipid mixturecomprises 70 mole-% of phosphatidylcholine and 30 mole-% ofphosphatidylserine. In other aspects, the phospholipid mixture comprises80 mole-% of phosphatidylcholine, 10 mole-% of phosphatidylserine, and10 mole-% of phosphatidylglycerol. In other aspects, the phospholipidmixture comprises 75 mole-% of phosphatidylcholine, 20 mole-% ofphosphatidylserine, and 5 mole-% of phosphatidylglycerol. In someaspects, the phospholipid mixture further comprises cholesterol. In someaspects, the cholesterol content in the phospholipid mixture is fromabout 1 to about 20 mole-% of cholesterol. In some aspects, thephospholipid mixture is in lipid vesicle form. In some aspects, thelipid vesicles are small unilamellar vesicles. In some aspects, theactivation mixture further comprises divalent cations. In other aspects,the divalent cations are calcium ions.

In some aspects, the activation mixture reacts with a coagulation factorselected from the group consisting of Factor VII, Factor VIII, andFactor IX. In other aspects, the Factor VIII coagulation factor is aFactor VIII protein or a fragment, variant, or derivative thereof. Insome aspects, the Factor IX coagulation factor is a Factor IX protein ora fragment, variant, or derivative thereof. In other aspects, the FactorVIII coagulation factor is a chimeric Factor VIII-Fc fusion protein. Insome aspects, the Factor IX coagulation factor is a chimeric FactorIX-Fc fusion protein. In other aspects, the Fc portion of the chimericFactor VIII or Factor IX protein comprises a human Fe domain. In someaspects, the chimeric Factor VIII protein comprises a B-domain deletedFactor VIII. In specific aspects, the chimeric Factor VIII proteincomprises SEQ ID NO:6. In other aspects, the chimeric Factor VIIIprotein comprises SEQ ID NO:2. In some aspects, the chimeric Factor IXprotein comprises SEQ ID NO: 13.

In some aspects, the solid substrate is selected from the groupconsisting of paper, plastic, glass, ceramic material, metal, andcombinations thereof. In other aspects, the solid substrate is a surfaceon a test strip, test stick, reaction chamber, cartridge, chip, wellplate, or array used in an apparatus to measure coagulation factoractivity or coagulation time. In some aspects, the patient has not yetbeen treated with a coagulation factor. In some aspects, the patient hasreceived prior coagulation factor treatment, but the treatment has beendiscontinued for a time period sufficient to deplete the coagulationfactor treatment from the patient's blood. In some aspects, themeasurement is carried in a point of care test system. In anotheraspect, the measurement is carried out in a mechanical or opticalanalytical system.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1 shows a schematic representation of the coagulation cascade.

FIG. 2 shows an overview of one stage assay (aPTT).

FIG. 3 shows clotting time for hemophilia A donor plasma spiked withrFVIIIFc measured using the Standard FMS Factor VIII assay.FIXa/phospholipid was used as activator mixture. Samples consisted of 12μL of re-calcified plasma applied directly (without preincubation) tothe test bed.

FIG. 4 shows clotting time for hemophilia B donor plasma spiked withrFIXIc measured using the Standard FMS Factor IX assay.FXIa/phospholipid was used as activator mixture. Samples consisted of 12μL of re-calcified plasma applied directly (without preincubation) tothe test bed.

FIG. 5A shows clotting time determined using the Standard FMS FactorVIII assay using Phospholipid Blend 2 in the activated coagulationfactor-phospholipid complex. Samples BD1-003. BD1-002, BD1-001 andBD1-005 were collected from 4 hemophilia A subjects and each sample wasspiked with 6 levels of rFVIIIFc (100%. 50%, 25%, 12.5%, 6.3% and 3.1%).

FIG. 5B shows clotting time determined using the Standard FMS FactorVIII assay using Phospholipid Blend 8 in the activated coagulationfactor-phospholipid complex. Samples BD1-003, BD1-002, BD1-001 andBD1-005 were collected from 4 hemophilia A subjects and each sample wasspiked with 6 levels of rFVIIIFc (100%, 50%, 25%, 12.5%, 6.3% and 3.1%).

FIG. 6A shows a comparison of clotting time determined using theStandard FMS Factor VIII assay (Standard Method) and Alternate FMSFactor VIII assay (Alternate Method 3). Samples contained 12 μL ofre-calcified plasma mixed 1:3 with substrate plasma (Factor VIIIdeficient plasma supplemented with defined levels of rFVIIIFc). FactorIXa/phospholipids complex was used as activator.

FIG. 6B shows clotting time measured using the Alternate FMS Factor IXassay (Alternate Method 9.8). Samples contained 12 μL of re-calcifiedplasma mixed 1:3 with substrate plasma (Factor IX deficient plasmasupplemented with defined levels of rFIXFc). Factor XIa/phospholipidscomplex was used as activator.

FIG. 7A shows the effect of a single plasma freeze-thaw cycle onAlternate FMS Factor IX assay performance. Samples contained 12 μL ofre-calcified plasma mixed 1:3 with substrate plasma (Factor IX deficientplasma supplemented with defined levels of rFIXFc).

FIG. 7B shows clotting times corresponding to fresh plasma samples, andplasma subjected to a single plasma freeze-thaw cycle as measured usingthe Alternate FMS Factor VIII assay. Samples contained 12 μL ofre-calcified plasma mixed 1:3 with substrate plasma (Factor VIIIdeficient plasma supplemented with defined levels of rFVIIIFc).

FIG. 8A shows the correlation between spiked rFVIIIFc levels measuredusing the Alternate FMS Factor VIII assay and rFVIIIFc levels measuredby MLA. Citrated plasma samples from 14 hemophilia A donor werecollected at 3 sites by 3 different methodologies, and spiked withvarying levels of rFVIIIFc prior to being assayed using the AlternateFMS Factor VIII assay or MLA.

FIG. 8B shows the correlation between spiked rFXFc levels measured usingthe Alternate FMS FIX assays and rFIXFc levels measured by MLA. Citratedplasma samples from 9 hemophilia B donor were collected at 3 sites using3 different methodologies, and spiked with varying levels of rFIXFcprior to being assayed using Alternate FMS FIX assay or MLA.

FIG. 9A shows clotting time results obtained by applying the StandardFMS FVIII assay to pre-dose whole blood samples obtained from twohemophilia A subjects (samples designated BD1-003 and BD1-005,respectively) spiked with increasing concentration of rFVIIIFc (0 IU/dlto 200 IU/dL).

FIG. 9B shows clotting time results obtained by applying the AlternateFMS FVIII assay to pre-dose whole blood samples obtained from twohemophilia A subjects (samples designated BD1-003 and BD1-005,respectively) spiked with increasing concentration of rFVIIIFc (0 IU/dlto 200 IU/dL).

FIGS. 10A, 10B and 10C show clotting times obtained using MLA assay.Samples were frozen plasma retains prepared from the samples spiked withvarious concentration of rFVIIFc. FIG. 10A shows results correspondingto Factor VIII calibration plasma and rFVIIIFc samples. FIG. 10B showsresults corresponding to pre-dose samples obtained from two hemophilia Asubjects (samples designated BD1-003 and BD1-005, respectively) spikedwith increasing concentration of rFVIIIFc. FIG. 10C shows resultscorresponding to post-dose samples obtained from two hemophilia Asubjects (samples designated BD1-003 and BD1-005, respectively) spikedwith increasing concentration of rFVIIIFc.

FIGS. 11A and 11B show, respectively, Standard FMS Factor VIII assaymeasurements conducted on frozen plasma retains of pre-dose (FIG. 11A)and post-dose (FIG. 11B) samples obtained from two hemophilia A subjects(samples designated BD1-003 and BD1-005, respectively) spiked withincreasing concentration of rFVIIIFc.

FIGS. 12A and 12B show, respectively. Alternate FMS Factor VIII assaymeasurements conducted on frozen plasma retains of pre-dose (FIG. 11A)and post-dose (FIG. 11B) samples obtained from two hemophilia A subjects(samples designated BD1-003 and BD1-005, respectively) spiked withincreasing concentration of rFVIIIFc.

FIG. 13 shows the correlation between whole blood clotting and plasmaclotting time measurements performed using the Alternate FMS FVIIIassay.

FIG. 14A compares Alternate FMS Factor VIII assay clotting times forplasma and whole blood samples obtained from two patients (BD1-002 andBD1-004). FIG. 14B shows the correlation between whole blood clottingtime and plasma clotting time measurements performed using the AlternateFMS FVIII assay. Pre-dose blood samples were spiked with increasingconcentrations of rFVIIIFc (0 IU/dL-200 IU/dL).

FIG. 15A compares Alternate FMS Factor IX assay clotting times forplasma and whole blood samples. FIG. 15B shows the correlation betweenwhole blood clotting time and plasma clotting time using the AlternateFMS FIX assay. Pre-dose blood samples were spiked with increasingconcentration of rFIXFc (0 IU/dL-200 IU/dL).

FIG. 16 shows the variability between meters assaying a single FVIIIdeficient plasma sample spiked to 100% (triangles) and 3% (circles)rFVIIIFc in duplicate on 16 research meters using the Alternate FMSFVIII assay.

FIG. 17 provides a diagram showing the correlation between coagulationfactor concentration (% Factor) and clotting time (Ct) from FMS assays,and the application of such correlation to a point-of-care device.

FIG. 18 provides a diagram showing the calculation of terminal half-lifefrom data obtained from FMS assays and its integration withpharmacokinetics data to calculate time to trough and determinepatient-specific doses and interval between doses.

DETAILED DESCRIPTION

The present disclosure provides methods and compositions for diagnosingand treating subject having a bleeding disorder. The disclosed methodscomprise contacting a sample. e.g., a blood sample or a plasma sampleobtained from the patient, with an activation mixture comprising anactivated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate. In some aspects, thetime between the contacting of the activation mixture with the bloodsample and the onset of clotting, i.e., the clotting time (Ct), is usedto calculate pharmacokinetic parameters which in turn can be used tocommence, modify, or cease treatment with coagulation factors. CertainFVIII and FIX polypeptides for use in the methods provided herein aredescribed in International Application No. PCT/US2010/059136, filed Dec.6, 2010, and in International Application No. PCT/US2011/043569, filedJul. 11, 2011, each of which is herein incorporated by reference in itsentirety.

I. Definitions

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a”, “an” and “the” include plural referentsunless the context clearly dictates otherwise. The terms “a” (or “an”),as well as the terms “one or more,” and “at least one” can be usedinterchangeably herein.

Furthermore, “and/or” where used herein is to be taken as specificdisclosure of each of the two specified features or components with orwithout the other. Thus, the term “and/or” as used in a phrase such as“A and/or B” herein is intended to include “A and B,” “A or B,” “A”(alone), and “B” (alone). Likewise, the term “and/or” as used in aphrase such as “A. B, and/or C” is intended to encompass each of thefollowing aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; Aand C; A and B; B and C; A (alone); B (alone); and C (alone).

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure is related. For example, the ConciseDictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed.,2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed.,1999, Academic Press; and the Oxford Dictionary Of Biochemistry AndMolecular Biology. Revised, 2000, Oxford University Press, provide oneof skill with a general dictionary of many of the terms used in thisdisclosure.

Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects ofthe disclosure, which can be had by reference to the specification as awhole. Accordingly, the terms defined immediately below are more fullydefined by reference to the specification in its entirety.

“Administering.” as used herein, refers to giving a pharmaceuticallyacceptable amount of a therapeutic agent such as a coagulation factor,e.g., Factor VIII or Factor IX polypeptide, to a subject via apharmaceutically acceptable route. Routes of administration includeintravenous, e.g., intravenous injection and intravenous infusion, e.g.,via central venous access. Additional routes of administration includesubcutaneous, intramuscular, oral, nasal, and pulmonary administration.In some aspects, the administration is subcutaneous. Coagulationfactors, e.g., Factor VIII and Factor IX, including fragments, variants,derivatives, chimeric polypeptides, or hybrid polypeptide can beadministered as part of a pharmaceutical composition comprising at leastone excipient. The term administering also refers to giving any othertherapeutic agent or prophylactic agent (e.g., a small molecule) thatcan be given in a pharmaceutically acceptable amount to a subject havinga coagulation-related disorder via a pharmaceutically acceptable route.

The term “sequence” as used to refer to a protein sequence, a peptidesequence, a polypeptide sequence, or an amino acid sequence means alinear representation of the amino acid constituents in the polypeptidein an amino-terminal to carboxyl-terminal direction in which residuesthat neighbor each other in the representation are contiguous in theprimary structure of the polypeptide.

By a “protein” or “polypeptide” is meant any sequence of two or moreamino acids linearly linked by amide bonds (peptide bonds) regardless oflength, post-translation modification, or function. As used herein, theterm “polypeptide” is intended to encompass a singular “polypeptide” aswell as plural “polypeptides.” “Polypeptide,” “peptide,” and “protein”are used interchangeably herein. Thus, peptides, dipeptides,tripeptides, or oligopeptides are included within the definition of“polypeptide.” and the term “polypeptide” can be used instead of, orinterchangeably with any of these terms. The term “polypeptide” is alsointended to refer to the products of post-expression modifications ofthe polypeptide, including without limitation glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, or modification bynon-naturally occurring amino acids. A polypeptide can be derived from anatural biological source or produced by recombinant technology, but isnot necessarily translated from a designated nucleic acid sequence. Apolypeptide can be generated in any manner, including by chemicalsynthesis. Also included as polypeptides of the present disclosure arefragments, derivatives, analogs, or variants of the foregoingpolypeptides, and any combination thereof.

The term “fragment” when referring to polypeptides and proteins, e.g.,coagulation factors such as Factor VIII or Factor IX, include anypolypeptides or proteins which retain at least some of the properties ofthe reference polypeptide or protein. E.g., in the case of procoagulantpolypeptides such as coagulation factors and procoagulant peptides, theterm fragment would refer to any polypeptides or proteins which retainat least some of the procoagulant activity of the reference polypeptideor protein. Fragments of polypeptides include proteolytic fragments, aswell as deletion fragments.

The term “variant” as used herein refers to a polypeptide sequence thatdiffers from that of a parent polypeptide sequence by virtue of at leastone amino acid modification. Variants can occur naturally or benon-naturally occurring. Non-naturally occurring variants can beproduced using art-known mutagenesis techniques. Variant polypeptidescan comprise conservative or non-conservative amino acid substitutions,deletions, or additions.

“Derivatives” of polypeptides or proteins of the present disclosure arepolypeptides or proteins which have been altered so as to exhibitadditional features not found on the native polypeptide or protein. Alsoincluded as “derivatives” are those peptides that contain one or morenaturally occurring amino acid derivatives of the twenty standard aminoacids. A polypeptide or amino acid sequence “derived from” a designatedpolypeptide or protein refers to the origin of the polypeptide.Preferably, the polypeptide or amino acid sequence which is derived froma particular sequence has an amino acid sequence that is essentiallyidentical to that sequence or a portion thereof, wherein the portionconsists of at least 10-20 amino acids, preferably at least 20-30 aminoacids, more preferably at least 30-50 amino acids, or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe sequence.

Polypeptides derived from another peptide can have one or more mutationsrelative to the starting polypeptide, e.g., one or more amino acidresidues which have been substituted with another amino acid residue orwhich has one or more amino acid residue insertions or deletions.Preferably, the polypeptide comprises an amino acid sequence which isnot naturally occurring. Such variants necessarily have less than 100%sequence identity or similarity with the starting polypeptide. In oneaspect, the variant will have an amino acid sequence from about 75% toless than 100% amino acid sequence identity or similarity with the aminoacid sequence of the starting polypeptide, more preferably from about80% to less than 100%, more preferably from about 85% to less than 100%,more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to lessthan 100%, e.g., over the length of the variant molecule. In one aspect,there is one amino acid difference between a starting polypeptidesequence and the sequence derived therefrom. Identity or similarity withrespect to this sequence is defined herein as the percentage of aminoacid residues in the candidate sequence that are identical (i.e. sameresidue) with the starting amino acid residues, after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity.

A polypeptide which is “isolated” is a polypeptide which is in a formnot found in nature. Isolated polypeptides include those which have beenpurified to a degree that they are no longer in a form in which they arefound in nature. In some aspects, a polypeptide which is isolated issubstantially pure.

A “recombinant” polypeptide or protein refers to a polypeptide orprotein produced via recombinant DNA technology. Recombinantly producedpolypeptides and proteins expressed in host cells are consideredisolated for the purpose of the invention, as are native or recombinantpolypeptides which have been separated, fractionated, or partially orsubstantially purified by any suitable technique. The polypeptidesdisclosed herein, e.g., clotting factors, can be recombinantly producedusing methods known in the art. Alternatively, proteins and peptidesdisclosed herein can be chemically synthesized.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art, including basic side chains (e.g., Lys, Arg,His), acidic side chains (e.g., Asp, Glu), uncharged polar side chains(e.g., Gly, Asn, Gnl, Ser, Thr, Tyr, Cys), nonpolar side chains (e.g.,Ala, Val, Leu, Ile, Pro, Phe, Met, Trp), beta-branched side chains(e.g., Thr, Val, Ile) and aromatic side chains (e.g., Tyr, Phe, Trp,His). Thus, if an amino acid in a polypeptide is replaced with anotheramino acid from the same side chain family, the substitution isconsidered to be conservative. In another aspect, a string of aminoacids can be conservatively replaced with a structurally similar stringthat differs in order and/or composition of side chain family members.

Non-conservative substitutions include those in which (i) a residuehaving an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, He, Phe or Val), (iii) a cysteineor proline is substituted for, or by, any other residue, or (iv) aresidue having a bulky hydrophobic or aromatic side chain (e.g., Val,He, Phe or Trp) is substituted for, or by, one having a smaller sidechain (e.g., Ala, Ser) or no side chain (e.g., Gly).

The term “percent sequence identity” between two polynucleotide orpolypeptide sequences refers to the number of identical matchedpositions shared by the sequences over a comparison window, taking intoaccount additions or deletions (i.e., gaps) that must be introduced foroptimal alignment of the two sequences. A matched position is anyposition where an identical nucleotide or amino acid is presented inboth the target and reference sequence. Gaps presented in the targetsequence are not counted since gaps are not nucleotides or amino acids.Likewise, gaps presented in the reference sequence are not counted sincetarget sequence nucleotides or amino acids are counted, not nucleotidesor amino acids from the reference sequence.

The percentage of sequence identity is calculated by determining thenumber of positions at which the identical amino-acid residue or nucleicacid base occurs in both sequences to yield the number of matchedpositions, dividing the number of matched positions by the total numberof positions in the window of comparison and multiplying the result by100 to yield the percentage of sequence identity. The comparison ofsequences and determination of percent sequence identity between twosequences can be accomplished using readily available software both foronline use and for download. Suitable software programs are availablefrom various sources, and for alignment of both protein and nucleotidesequences. One suitable program to determine percent sequence identityis bl2seq, part of the BLAST suite of program available from the U.S.government's National Center for Biotechnology Information BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq performs a comparison between twosequences using either the BLASTN or BLASTP algorithm. BLASTN is used tocompare nucleic acid sequences, while BLASTP is used to compare aminoacid sequences. Other suitable programs are, e.g., Needle, Stretcher,Water, or Matcher, part of the EMBOSS suite of bioinformatics programsand also available from the European Bioinformatics Institute (EBI) atwww.ebi.ac.uk/Tools/psa.

Different regions within a single polynucleotide or polypeptide targetsequence that aligns with a polynucleotide or polypeptide referencesequence can each have their own percent sequence identity. It is notedthat the percent sequence identity value is rounded to the nearesttenth. For example, 80.11, 80.12, 80.13, and 80.14 are rounded down to80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to80.2. It also is noted that the length value will always be an integer.

In certain aspects, the percentage identity “X” of a first amino acidsequence to a second sequence amino acid is calculated as 100×(Y/Z),where Y is the number of amino acid residues scored as identical matchesin the alignment of the first and second sequences (as aligned by visualinspection or a particular sequence alignment program) and Z is thetotal number of residues in the second sequence. If the length of afirst sequence is longer than the second sequence, the percent identityof the first sequence to the second sequence will be higher than thepercent identity of the second sequence to the first sequence.

One skilled in the art will appreciate that the generation of a sequencealignment for the calculation of a percent sequence identity is notlimited to binary sequence-sequence comparisons exclusively driven byprimary sequence data. Sequence alignments can be derived from multiplesequence alignments. One suitable program to generate multiple sequencealignments is ClustalW2, available from www.clustal.org. Anothersuitable program is MUSCLE, available from www.drive5.com/muscle/.ClustalW2 and MUSCLE are alternatively available, e.g., from the EBI.

It will also be appreciated that sequence alignments can be generated byintegrating sequence data with data from heterogeneous sources such asstructural data (e.g., crystallographic protein structures), functionaldata (e.g., location of mutations), or phylogenetic data. A suitableprogram that integrates heterogeneous data to generate a multiplesequence alignment is T-Coffee, available at www.tcoffee.org, andalternatively available, e.g., from the EBI. It will also be appreciatedthat the final alignment used to calculate percent sequence identity canbe curated either automatically or manually.

“Polynucleotide” and “nucleic acid” are used interchangeably and referto a polymeric compound comprised of covalently linked nucleotideresidues. Polynucleotides can be DNA, cDNA, RNA, single stranded, ordouble stranded, vectors, plasmids, phage, or viruses. Polynucleotidesinclude those in Sequence Table 1, which encode the polypeptides ofSequence Table 2 (see Sequence Table 1). Polynucleotides also includefragments of the polynucleotides of Table 1, e.g., those that encodefragments of the polypeptides of Table 2, such as Factor VIII, FactorIX, Fc, signal sequence, propeptide, 6His and other fragments of thepolypeptides of Sequence Table 2.

The terms “subject” and “patient” are used interchangeably and refer toa human or a non-human mammal, for whom diagnosis, prognosis, or therapyof a bleeding disorder is desired. Non-human mammals include mice, dogs,primates, bears, cats, horses, cows, pigs, and other domestic animalsand small animals. Subjects also include pediatric humans. Pediatrichuman subjects are birth to 20 years, e.g., birth to 18 years, birth to16 years, birth to 15 years, birth to 12 years, birth to 11 years, birthto 6 years, birth to 5 years, birth to 2 years, or 2 to 11 years of age.In some aspects of the present disclosure, a subject is a naïve subject.A naïve subject is a subject that has not been administered a treatmentfor a bleeding disorder. In some aspects, a naïve subject has not beentreated with prior to being diagnosed with having a bleeding disorder.

The methods disclosed herein can be practiced on a subject in need ofcontrol or prevention of bleeding, bleeding episodes, or hemophiliadisorders. Such subjects include those in need of control or preventionof bleeding in minor hemorrhage, hemarthroses, superficial musclehemorrhage, soft tissue hemorrhage, moderate hemorrhage, intramuscle orsoft tissue hemorrhage with dissection, mucous membrane hemorrhage,hematuria, major hemorrhage, hemorrhage of the pharynx, hemorrhage ofthe retropharynx, hemorrhage of the retroperitonium, hemorrhage of thecentral nervous system, bruises, cuts, scrapes, joint hemorrhage, nosebleed, mouth bleed, gum bleed, intracranial bleeding, intraperitonealbleeding, minor spontaneous hemorrhage, bleeding after major trauma,moderate skin bruising, or spontaneous hemorrhage into joints, muscles,internal organs or the brain. Such subjects also include those need ofperi-operative management, such as management of bleeding associatedwith surgery or dental extraction.

The term “bleeding disease or disorder.” as used herein, means agenetically inherited or acquired condition characterized by a tendencyto hemorrhage, either spontaneously or as a result of trauma, due to animpaired ability or inability to form a fibrin clot. Examples of suchdisorders include hemophilias. The three main forms are hemophilia A(factor VIII deficiency), hemophilia B (factor IX deficiency or“Christmas disease”) and hemophilia C (factor XI deficiency, mildbleeding tendency). Other hemostatic disorders include, e.g., vonWillebrand disease, Factor XI deficiency (PTA deficiency). Factor XIIdeficiency, deficiencies or structural abnormalities in fibrinogen,prothrombin, Factor V, Factor VII, Factor X or factor XIII,Bernard-Soulier syndrome, which is a defect or deficiency in GPIb. GPIb,the receptor for vWF, can be defective and lead to lack of primary clotformation (primary hemostasis) and increased bleeding tendency), andthrombasthenia of Glanzman and Naegeli (Glanzmann thrombasthenia). Inliver failure (acute and chronic forms), there is insufficientproduction of coagulation factors by the liver; this can increasebleeding risk.

Bleeding disease or disorder can require on-demand treatment orprophylactic treatment. “On-demand treatment,” as used herein, meanstreatment that is intended to take place over a short course of time andis in response to an existing condition, such as a bleeding episode, ora perceived short term need such as planned surgery. Conditions that canrequire on-demand treatment include a bleeding episode, hemarthrosis,muscle bleed, oral bleed, hemorrhage, hemorrhage into muscles, oralhemorrhage, trauma, trauma capitis, gastrointestinal bleeding,intracranial hemorrhage, intra-abdominal hemorrhage, intrathoracichemorrhage, bone fracture, central nervous system bleeding, bleeding inthe retropharyngeal space, bleeding in the retroperitoneal space, orbleeding in the illiopsoas sheath. Bleeding episodes other than theseare also included. The subject can be in need of surgical prophylaxis,peri-operative management, or treatment for surgery. Such surgeriesinclude minor surgery, major surgery, tooth extraction, tonsillectomy,other dental/thoraco-facial surgeries, inguinal herniotomy, synovectomy,total knee replacement, other joint replacement, craniotomy,osteosynthesis, trauma surgery, intracranial surgery, intra-abdominalsurgery, intrathoracic surgery. Surgeries other than these are alsoincluded.

Additional conditions that can require on-demand treatment include minorhemorrhage, hemarthroses, superficial muscle hemorrhage, soft tissuehemorrhage, moderate hemorrhage, intramuscle or soft tissue hemorrhagewith dissection, mucous membrane hemorrhage, hematuria, majorhemorrhage, hemorrhage of the pharynx, hemorrhage of the retropharynx,hemorrhage of the retroperitonium, hemorrhage of the central nervoussystem, bruises, cuts, scrapes, joint hemorrhage, nose bleed, mouthbleed, gum bleed, intracranial bleeding, intraperitoneal bleeding, minorspontaneous hemorrhage, bleeding after major trauma, moderate skinbruising, or spontaneous hemorrhage into joints, muscles, internalorgans or the brain. Additional reasons for on-demand treatment includethe need for peri-operative management for surgery or dental extraction,major surgery, extensive oral surgery, urologic surgery, hernia surgery,orthopedic surgery such as replacement of knee, hip, or other majorjoint.

The terms “prophylactic treatment” or “prophylaxis” as used herein, meanadministering a procoagulant compound, e.g., a clotting factor,fragment, variant, derivative, chimeric peptide, or hybrid peptidethereof, to a subject over a course of time to increase the level ofactivity in a subject's plasma. Preferably, the increased level issufficient to decrease the incidence of spontaneous bleeding or toprevent bleeding, e.g., in the event of an unforeseen injury.Preferably, during prophylactic treatment, the plasma protein level inthe subject does not fall below the baseline level for that subject, orbelow the level that characterizes severe hemophilia.

The term “about” is used herein to mean approximately, roughly, around,or in the regions of. When the term “about” is used in conjunction witha numerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. Thus, “about 10-20”means “about 10 to about 20.” In general, the term “about” is usedherein to modify a numerical value above and below the stated value by avariance of 10 percent, up or down (higher or lower).

The term “pharmacokinetic parameters” or “PK parameters” as used hereinrefers to those constant and variable terms that are related to thedisposition of a pharmacologically active agent. e.g., a coagulationfactor, within a subject and includes for example volume ofdistribution, total clearance, metabolic clearance, bioavailability,intrinsic clearance, mean residence time, partitioning coefficientsbetween tissues and blood, elimination rates, half-life, terminalhalf-life, time to trough, as well as other parameters known in the art.PK parameters can be based, e.g., on protein level or activity level. Inaddition, certain PK parameters can be based on model predicted data, onobserved data, or on combinations of model and observed data.

As used herein, the term “clotting factor.” refers to molecules,fragment, derivatives, or analogs thereof, naturally occurring orrecombinantly produced, which prevent or decrease the duration of ableeding episode in a subject. In other words, it means molecules havingpro-clotting or pro-coagulant activity, i.e., are responsible for theconversion of fibrinogen into a mesh of insoluble fibrin causing theblood to coagulate or clot. The term “clotting factor” as used hereinalso encompasses synthetic peptides with procoagulant activity.

The term “clotting time” as used herein, refers to the time periodelapsed from the time when the sample is contacted with the activatingmixture until the time when the sample clots.

“Half-Life” as used herein, refers to a biological half-life of aparticular therapeutic agent in vivo. Terminal half-life can berepresented by the time required for half the quantity administered to asubject to be cleared from the circulation and/or other tissues in thesubject. When a clearance curve of a given polypeptide is constructed asa function of time, the curve is usually biphasic with a rapid α-phaseand longer β-phase. The α-phase typically represents an equilibration ofthe administered chimeric polypeptide between the intra- andextra-vascular space and is, in part, determined by the size of thepolypeptide. The β-phase typically represents the catabolism of thepolypeptide in the intravascular space.

The term “terminal plasma half-life” or “terminal half-life” refers tothe time required to divide the plasma concentration by two afterreaching pseudo-equilibrium. The terminal half-life is especiallyrelevant to multiple dosing regimens, because it controls the degree oftherapeutic agent accumulation, concentration fluctuations, and the timetaken to reach equilibrium.

“Trough,” as used herein, is the lowest plasma activity level reachedafter administering a dose of a pharmacologically active agent, e.g., aclotting factor such as Factor VIII or Factor IX, a fragment, aderivative or an analog thereof, before the next dose is administered,if any. Accordingly. “time to trough” (T) is the time at which thelowest plasma activity level is reached after administering apharmacologically active agent before the next dose is administered.

The term “sample” as used herein includes any biological fluid or issue,such as whole blood or serum, obtained from a subject which contains oris suspected to contain a blood coagulation factor. In some specificaspects, that sample is blood or a fraction thereof, muscle, skin, or acombination thereof. Samples can be obtained by any means known in theart.

In order to apply the methods and systems of the disclosure, samplesfrom a patient can be obtained before or after the administration of atherapy to treat a bleeding disorder. In some cases, successive samplescan be obtained from the patient after therapy has commenced or aftertherapy has ceased. Samples can, for example, be requested by ahealthcare provider (e.g., a doctor) or healthcare benefits provider,obtained and/or processed by the same or a different healthcare provider(e.g., a nurse, a hospital) or a clinical laboratory, and afterprocessing, the results can be forwarded to yet another healthcareprovider, healthcare benefits provider or the patient. Similarly, themeasuring/determination of clotting times and/or PK parameters derivedfrom clotting times, comparisons between clotting times and/or PKparameters derived from clotting times, evaluation of the clotting timesand/or PK parameters derived from clotting time, and treatment decisionscan be performed by one or more healthcare providers, healthcarebenefits providers, and/or clinical laboratories.

As used herein, the term “healthcare provider” refers to individuals orinstitutions which directly interact and administer to living subjects.e.g., human patients. Non-limiting examples of healthcare providersinclude doctors, nurses, technicians, therapist, pharmacists,counselors, alternative medicine practitioners, medical facilities,doctor's offices, hospitals, emergency rooms, clinics, urgent carecenters, alternative medicine clinics/facilities, and any other entityproviding general and/or specialized treatment, assessment, maintenance,therapy, medication, and/or advice relating to all, or any portion of, apatient's state of health, including but not limited to general medical,specialized medical, surgical, and/or any other type of treatment,assessment, maintenance, therapy, medication and/or advice.

As used herein, the term “clinical laboratory” refers to a facility forthe examination or processing of materials derived from a livingsubject, e.g., a human being. Non-limiting examples of processinginclude biological, biochemical, serological, chemical,immunohematological, hematological, biophysical, cytological,pathological, genetic, or other examination of materials derived fromthe human body for the purpose of providing information, e.g., for thediagnosis, prevention, or treatment of any disease or impairment of, orthe assessment of the health of living subjects, e.g., human beings.These examinations can also include procedures to collect or otherwiseobtain a sample, prepare, determine, measure, or otherwise describe thepresence or absence of various substances in the body of a livingsubject, e.g., a human being, or a sample obtained from the body of aliving subject. e.g., a human being.

As used herein, the term “healthcare benefits provider” encompassesindividual parties, organizations, or groups providing, presenting,offering, paying for in whole or in part, or being otherwise associatedwith giving a patient access to one or more healthcare benefits, benefitplans, health insurance, and/or healthcare expense account programs.

In some aspects, a healthcare provider can administer or instructanother healthcare provider to administer a therapy to treat a bleedingdisease or disorder. A healthcare provider can implement or instructanother healthcare provider or patient to perform the following actions:obtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples, administer a therapy ortherapeutic agent (e.g., a clotting factor such as a Factor VIII orFactor IX polypeptide), commence the administration of a therapy, ceasethe administration of a therapy, continue the administration of atherapy, temporarily interrupt the administration of a therapy, increasethe amount of an administered therapeutic agent, decrease the amount ofan administered therapeutic agent, continue the administration of anamount of a therapeutic agent, increase the frequency of administrationof a therapeutic agent, decrease the frequency of administration of atherapeutic agent, maintain the same dosing frequency on a therapeuticagent, replace a therapy or therapeutic agent by at least anothertherapy or therapeutic agent, combine a therapy or therapeutic agentwith at least another therapy or additional therapeutic agent.

In some aspects, a healthcare benefits provider can authorize or deny,for example, collection of a sample, processing of a sample, submissionof a sample, receipt of a sample, transfer of a sample, analysis ormeasurement a sample, quantification a sample, provision of resultsobtained after analyzing/measuring/quantifying a sample, transfer ofresults obtained after analyzing/measuring/quantifying a sample,comparison/scoring of results obtained afteranalyzing/measuring/quantifying one or more samples, transfer of thecomparison/score from one or more samples, administration of a therapyor therapeutic agent, commencement of the administration of a therapy ortherapeutic agent, cessation of the administration of a therapy ortherapeutic agent, continuation of the administration of a therapy ortherapeutic agent, temporary interruption of the administration of atherapy or therapeutic agent, increase of the amount of administeredtherapeutic agent, decrease of the amount of administered therapeuticagent, continuation of the administration of an amount of a therapeuticagent, increase in the frequency of administration of a therapeuticagent, decrease in the frequency of administration of a therapeuticagent, maintain the same dosing frequency on a therapeutic agent,replace a therapy or therapeutic agent by at least another therapy ortherapeutic agent, or combine a therapy or therapeutic agent with atleast another therapy or additional therapeutic agent.

In addition a healthcare benefits providers can, e.g., authorize or denythe prescription of a therapy, authorize or deny coverage for therapy,authorize or deny reimbursement for the cost of therapy, determine ordeny eligibility for therapy, etc.

In some aspects, a clinical laboratory can, for example, collect orobtain a sample, process a sample, submit a sample, receive a sample,transfer a sample, analyze or measure a sample, quantify a sample,provide the results obtained after analyzing/measuring/quantifying asample, receive the results obtained afteranalyzing/measuring/quantifying a sample, compare/score the resultsobtained after analyzing/measuring/quantifying one or more samples,provide the comparison/score from one or more samples, obtain thecomparison/score from one or more samples.

The above enumerated actions can be performed by a healthcare provider,healthcare benefits provider, or patient automatically using acomputer-implemented method (e.g., via a web service or stand-alonecomputer system).

II. Methods and Compositions for Coagulation Activity Testing

The standard methodology for determining coagulation factor levels inuse today is the one stage coagulation factor clotting assay (FIG. 2). Amajor drawback of this assay is that one cannot use whole blood samples.A typical one stage coagulation factor clotting assay requires, forexample (i) venous blood drawn into sodium citrate anticoagulant, (ii)centrifugation to obtain a plasma sample, (iii) laboratory bench topcoagulation analyzers and specifically trained laboratory personnel,(iv) liquid reagent preparation and standard curve construction, (v)multiple step assay procedures, (vi) dilution of patient plasma, (vii)mix with factor deficient plasma to mask inter-individual phenotypicvariation, (viii) pre-incubation with non-physiological contact phaseactivators to generate an activated factor, e.g., Factor XIa, (ix)addition of Ca⁺² to initiate clotting, (x) optical (most common) ormechanical clot detection, (xi) derivation of factor level fromLog-Linear (most common) plot of coagulation factor concentration versusclot time (reliable range for standard instrument systems usinglog-linear fits is ˜3%-120% of normal; high end instruments haveincorporated sophisticated software packages that can return accuratevalues below 1%).

To address the drawbacks of conventional one stage coagulation factorclotting assays, the present disclosure provides a modified coagulationassay which, in contrast with a standard coagulation assay, can operateusing a whole blood sample, for example, fingerstick blood. Instead ofpre-incubating the sample with a non-physiological contact phaseactivators. e.g., kaolin, typically used in laboratory-based assays, theassays disclosed herein use an activation mixture comprising anactivated coagulation factor-phospholipid complex. This activationmixture is dried onto a solid substrate, e.g., a test strip.

Accordingly, the disclosed assays can be performed in point of careanalyzers that do not require specially trained laboratory personnel.This general assay format, in which a patient sample (plasma or wholeblood) can be applied directly to the solid substrate containing driedassay chemistry, is referred to as the Standard Factor Monitoring System(“Standard FMS”) assay throughout the present disclosure.

In specific aspects of the present disclosure, the Standard FMS assayscan be applied to determining coagulation activity of Factor VIII, e.g.,measured as clotting time. For the Standard FMS Factor VIII assay, theactivated coagulation factor-phospholipid complex can comprise, forexample, a mixture of purified activated Factor IX (Factor IXa;abbreviated as FIXa) and phospholipid vesicles, wherein the activationmixture is dried onto a solid substrate. In other specific aspects ofthe present disclosure, the Standard FMS assay can be applied todetermining coagulation activity of Factor IX. e.g., measured asclotting time. For the Standard FMS Factor IX assay, the activatedcoagulation factor-phospholipid complex can comprise, for example, amixture of purified activated Factor XI (Factor XIa; abbreviated asFXIa) and phospholipid vesicles, wherein the activation mixture is driedonto a solid substrate. In some aspects, the assays disclosed hereinrequire no preincubation of the samples with the activators mixture.

In order to address the observed phenotypic variability between samplesfrom the same donor or between donors, the Standard FMS assay can bemodified. In some aspects of the present disclosure, less sensitivephospholipid blends in the activator mixture can be used to reducephenotypic variability. In other aspects, adding a variety of purifiedcoagulation factors to the sample. e.g., Factor II, Factor VII. FactorVIII. Factor IX. Factor X. Factor XI. Factor XII. Factor XIII,fibrinogen, vWF, or Tissue Factor can also reduce phenotypicvariability. In other aspects, adding inhibitors to the sample. e.g.,CTI, aprotinin, ϵ-aminocaproic acid (EACA).D-Phenylalanyl-1-prolyl-1-arginine chloromethyl ketone-Factor VIIa(FPRCK-FVIIa), or anti-FVIII monoclonal antibodies can also reducephenotypic variability. Accordingly, the present disclosure providesalso a variant of the Standard FMS assay, referred to as the “AlternateFMS” assay throughout the instant disclosure. This Alternate FMS assayis essentially a hybrid between the Standard FMS assay and a one stagefactor assay (e.g., an aPTT assay) which is less susceptible tophenotypic variability. The Alternate FMS assay also utilizes anactivation mixture comprising a coagulation factor (e.g., FIXa or FXIa)and a phospholipid vesicle preparation dried on the solid substrate(e.g. a disposable test strip). In the plasma based Alternate FMS assay,one part of sample (e.g., hemophilia plasma or fingerstick whole blood)can be mixed with a volume of a corresponding sample that has beendepleted of the assay target factor (referred to as “substrate sample”throughout the instant disclosure). In this manner, the variability ofnon-target sample components can be normalized by addition of thesubstrate sample. This combination of sample (e.g., hemophilia plasma orfingerstick whole blood) and substrate sample can be done in anall-liquid system, resulting in a dilution of the sample, thusincreasing the lower level of detection of the assay. In some aspects,the sample is diluted with substrate sample at about a 1:2 ratio, atabout 1:3 ratio, at about a 1:4 ratio, or at about a 1:5 ratio. Dilutionratios can be adjusted above or below the disclosed ratios using routineexperimentation.

As disclosed above, both the Standard FMS assay and the Alternate FMSassays use an activation mixture comprising an activated coagulationfactor and a phospholipid mixture, wherein the composition is dried ontoa solid substrate. The activation mixture can contain all the substancesnecessary for the determination of coagulation factor activity, e.g.,via measurement of clotting time. These necessary substances are usuallyan activated coagulation factor functioning as coagulation activatorcomponent, phospholipids, and optionally divalent cations.

The activation mixtures disclosed herein generally do not containstabilizers, however, in some aspects of the present disclosure, theactivation mixture can contain one or more stabilizers known in the art,such as amino acids (e.g., D-alanine, L-alanine, beta-alanine, etc.).Suitable concentrations of stabilizers are known in the art or can beroutinely determined. In some specific aspects, the activation mixturedisclosed herein consists of or substantially consists of an activatedcoagulation factor and a phospholipid mixture, i.e., the activationmixture it does not contain, e.g., divalent cations, stabilizers such asalbumin o amino acids, or additional coagulation factor activators orinhibitors.

In some aspects, the solid substrate can be, e.g., paper, plastic,glass, ceramic material, metal, and combinations thereof. The solidsubstrate can be, for example, the surface on a test strip, test stick,reaction chamber, cartridge, chip, well plate, or array used in anapparatus to measure coagulation factor activity or coagulation time. Insome aspects, the solid substrate can be a membrane, which can be singlelayered or multilayered. In some aspects, the solid substrate is thesurface of a disposable test strip. In other aspects, the solidsubstrate is the wall in a well in a plastic cartridge. In otheraspects, the solid substrate is the wall of a well in a multiwell plate(e.g., a 96-well place). In some aspects, the solid substrate is thewall of a capillary. In other aspects, the solid substrate is the wallof a vial. In other aspects, the solid substrate is a surface in amechanical mixing component of a measurement apparatus.

The solid substrate can be made of any suitable material whichpreferably has good thermal conductivity, clarity for opticaltransmission, mechanical properties for easy construction, surfaceproperties that allow for uniform drying and stability of the activationmixture, and neutrality to the liquid medium in the sample to preventinterference with the coagulation assay. For this purpose, plastic areespecially well suited. Suitable plastics include, for example, thosewith high free surface energies and low water sorption, including PETG,polyester (MYLAR®), polycarbonate (LEXAN®), polyvinyl chloride,polystyrene. SAN, acrylonitrile-butadiene-styrene (ABS) (e.g.,CYCOLAC®), etc. In some aspects, plastics and other materials used assolid substrates can be hydrophobic, which would make it difficult touniformly coat the surface with the activation mixtures disclosed here.Therefore, in some aspects, the substrate can be coated with anotherreagent (e.g., chemicals such aspoly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx),poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), or polylactic acid(PLA); or proteins such as collagen or fibronectin) that would renderthe surface of the substrate hydrophilic and permit attachment of theactivation mixture to the surface. In other aspects, the substrate canbe physically modified by plasma etching or corona treating to renderits surface hydrophilic.

The activation mixture can be provided, for example, (i) already driedonto a solid substrate, (ii) in a liquid to be dried in situ, or (iii)in a dry form (e.g., lyophilized form) to be reconstituted and driedonto the solid substrate. Dry components can be provided separately orin a premixed form. The activation mixture can be dried onto the solidsubstrate by using methods known in the art. For example, the drying ofthe activation mixture can be accomplished by air drying (e.g., at roomtemperature), drying under an inert gas stream (e.g., nitrogen orargon), vacuum drying, lyophilizing, dessicant drying, convectivedrying, etc. The term drying “onto” a solid substrate also encompassesdrying the activation mixture “into” a porous substrate. In thisrespect, the dry activation mixture can be, for example, located intoporous matrices such as sponges, porous paper filters, fleece or feltmaterial, or can be microencapsulated.

The activation mixture can be applied to the substrate using methodsknown in the art, e.g., spray painting or lyophilization. In someaspects, the activation mixture can be chemically conjugated to thesubstrate. Chemical conjugation methods to covalently attach lipids,e.g., phospholipids and/or proteins, e.g., coagulation factors, areknown in the art.

In some aspects, the activation mixture disclosed herein can be used tomeasure clotting time in samples containing or suspected to contain acoagulation factor, for example. Factor VIII or Factor IX. In someaspects, the coagulation factor is a Factor VIII protein or a fragment,variant, or derivative thereof as disclosed below. In other aspects, thecoagulation factor is a Factor IX protein or a fragment, variant, orderivative thereof. In some specific aspects, the Factor VIII or FactorIX proteins are chimeric proteins (e.g., rFVIIIFc or rFIXFc) or hybridproteins.

In some aspects, the Factor VIII chimeric protein is a single chain (SC)rFVIIIFc. SC rFVIIIFc are disclosed, for example, in U.S. ProvisionalApplication No. 61/668,889, and U.S. Pat. No. 7,041,635, both of whichare herein incorporated by reference in their entireties.

The activation mixture disclosed herein can contain an activatedcoagulation factor, or alternatively a hematologically equivalent, such,as a fragment, variant, or derivative thereof.

In some aspects, for example to apply the methods disclosed herein tomeasure the coagulation activity of a Factor VIII protein (or afragment, variant, derivative, chimeric protein or hybrid proteinthereof), the activated coagulation factor is a Factor IXa protein or afragment, variant, or derivative thereof. In some specific aspects, aFactor IXa protein (or a fragment, variant, derivative, chimeric proteinor hybrid protein thereof) is present in the activation mixturecomposition prior to drying within a range of about 0.01 U/mL to about0.05 U/mL. In some aspects, the concentration of Factor IXa protein or afragment, variant, or derivative thereof is about 0.01 U/mL about 0.02U/mL, about 0.03 U/mL, about 0.04 U/mL, about 0.05 U/mL, about 0.06U/mL, about 0.07 U/mL, about 0.08 U/mL, about 0.09 U/mL, or about 0.1U/mL. In some aspects, the concentration of Factor IXa protein or afragment, variant, or derivative thereof is at least about 0.1 U/mL.

In other aspects, for example to apply the methods disclosed herein tomeasure the coagulation activity of a Factor IX protein or a fragment,variant, or derivative thereof, the activated coagulation factor is aFactor XIa protein or a fragment, variant, or derivative thereof. Insome specific aspects, the Factor XIa protein or a fragment, variant, orderivative thereof is present in the activation mixture compositionprior to drying within a range of about 0.01 U/mL to about 0.05 U/mL. Insome aspects, the concentration of Factor XIa protein or a fragment,variant, or derivative thereof is about 0.01 U/mL, about 0.02 U/mL,about 0.03 U/mL, about 0.04 U/mL, about 0.05 U/mL, about 0.06 U/mL,about 0.07 U/mL, about 0.08 U/mL, about 0.09 U/mL, or about 0.1 U/mL. Insome aspects, the concentration of Factor XIa protein or a fragment,variant, or derivative thereof is at least 0.1 U/mL. In some aspects,the concentration of Factor XIa protein or a fragment, variant, orderivative thereof is about 0.01 mg/mL, about 0.02 mg/mL, about 0.03mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06.g/mL, about 0.07mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, or about 0.1 mg/mL. In someaspects, the concentration of Factor XIa protein or a fragment, variant,or derivative thereof is about 0.1 mg/mL. In some aspects, theconcentration of Factor XIa protein or a fragment, variant, orderivative thereof is about 0.10 mg/mL, about 0.15 mg/mL, about 0.20mg/mL, about 0.25 mg/mL, about 0.30 mg/mL, about 0.35.g/mL, about 0.40mg/mL, about 0.45 mg/mL, or about 0.50 mg/mL.

One skilled in the art would understand that other activated coagulationfactors and cofactors can be used instead of Factor IXa and FXIadepending on the coagulation factor tested in the coagulation assay.

The activation mixture contains a phospholipid mixture comprising atleast one phospholipid. In some aspects, the phospholipid mixturecomprises 2 phospholipids. In other aspects, the phospholipid mixturecomprises 3 phospholipids. In other aspects, the phospholipid mixturecomprises more than three phospholipids. In other aspects, thephospholipid mixture comprises at least one phospholipid in combinationwith at least another lipid, e.g., a fatty acid or cholesterol.

In some specific aspects, the composition of the phospholipid mixture isdefined. i.e., phospholipid(s) and other lipid components (if present)are combined according to predetermined ratios. In other aspects, thecomposition of phospholipid mixture is not defined. e.g., thephospholipid mixture is obtained from an animal and/or vegetal tissueextract (e.g., egg, soy, etc). Chloroform extracts from rabbit brain arean example of suitable phospholipid mixture obtained from a tissueextract known in the art. In some aspects, the phospholipids can benatural. In other aspects, the phospholipids can be synthetic. In someaspects, the phospholipids are a mixture of natural and syntheticphospholipids. The phospholipids in the phospholipid mixture can be, forexample, phosphatidylcholine, phosphatidylserine, phosphatidylglycerol,phosphatidic acid, phosphatidylethanolamine, and any combinationsthereof.

Synthetic phospholipics that can be present in the phospholipid mixtureinclude, for example, synthetic phosphatidic acid (e.g., DMPA, DPPA,DSPA), synthetic phosphatidylcholine (e.g., DDPC, DLPC, DMPC. DPPC.DSPC, DOPC, POPC, DEPC), synthetic phosphatidylglycerol (e.g., DMPG,DPPG, DSPG, POPG), synthetic phosphatidylethanoamine (e.g., DMPE, DPPE,DSPE, DOPE), synthetic phosphatidylserine (e.g., DOPS), and combinationsthereof.

In some specific examples, the phospholipid mixture comprises 70 mole-%of phosphatidylcholine and 30 mole-% of phosphatidylserine. In otherspecific examples, the phospholipid mixture comprises 80 mole-% ofphosphatidylcholine, 10 mole-% of phosphatidylserine, and 10 mole-% ofphosphatidylglycerol. In yet other specific examples, the phospholipidmixture comprises 75 mole-% of phosphatidylcholine, 20 mole-% ofphosphatidylserine, and 5 mole-% of phosphatidylglycerol.

In some specific examples, the phospholipid mixture consists or consistsessentially of 70 mole-% of phosphatidylcholine and 30 mole-% ofphosphatidylserine. In other specific examples, the phospholipid mixtureconsists or consists essentially of 80 mole-% of phosphatidylcholine, 10mole-% of phosphatidylserine, and 10 mole-% of phosphatidylglycerol. Inyet other specific examples, the phospholipid mixture consists orconsists essentially of 75 mole-% of phosphatidylcholine, 20 mole-% ofphosphatidylserine, and 5 mole-% of phosphatidylglycerol.

In some aspects, the phospholipid mixture comprises at least about 5mole-%, at least about 10 mole-%, at least about 15 mole-%, at leastabout 20 mole-%, at least about 25 mole-%, at least about 30 mole-%, atleast about 35 mole-%, at least about 40 mole-%, at least about 45mole-%, at least about 50 mole-%, at least about 55 mole-%, at leastabout 60 mole-%, at least about 65 mole-%, at least about 70 mole-%, atleast about 75 mole-%, at least about 80 mole-%, at least about 85mole-%, at least about 90 mole-%, or at least about 95 mole-% ofphosphatidylcholine.

In some aspects, the phospholipid mixture comprises at least about 5mole-%, at least about 10 mole-%, at least about 15 mole-%, at leastabout 20 mole-%, at least about 25 mole-%, at least about 30 mole-%, atleast about 35 mole-%, at least about 40 mole-%, at least about 45mole-%, at least about 50 mole-%, at least about 55 mole-%, at leastabout 60 mole-%, at least about 65 mole-%, at least about 70 mole-%, atleast about 75 mole-%, at least about 80 mole-%, at least about 85mole-%, at least about 90 mole-%, or at least about 95 mole-% ofphosphatidylserine.

In some aspects, the phospholipid mixture comprises at least about 5mole-%, at least about 10 mole-%, at least about 15 mole-%, at leastabout 20 mole-%, at least about 25 mole-%, at least about 30 mole-%, atleast about 35 mole-%, at least about 40 mole-%, at least about 45mole-%, at least about 50 mole-%, at least about 55 mole-%, at leastabout 60 mole-%, at least about 65 mole-%, at least about 70 mole-%, atleast about 75 mole-%, at least about 80 mole-%, at least about 85mole-%, at least about 90 mole-%, or at least about 95 mole-% ofphosphatidylglycerol.

In some aspects, the phospholipid mixture further comprises cholesterol.In some aspects, the phospholipid mixture comprises at least about 1mole-%, at least about 2 mole-%, at least about 3 mole-%, at least about4 mole-%, at least about 5 mole-%, at least about 6 mole-%, at leastabout 7 mole-%, at least about 8 mole-%, at least about 9 mole-%, atleast about 10 mole-%, at least about 11 mole-%, at least about 12mole-%, at least about 13 mole-%, at least about 14 mole-%, at leastabout 15 mole-%, at least about 16 mole-%, at least about 17 mole-%, atleast about 18 mole-%, at least about 19 mole-%, or at least about 20mole-% of cholesterol.

In some aspects, the phospholipid mixture is combined with the activatedcoagulation factor prior to drying onto a solid substrate. In someaspects, the phospholipid mixture is in vesicle form (e.g., a liposomeor other artificial lipid vesicle). In some aspects, the vesicles areunilamellar vesicles, e.g., small unilamellar vesicles. Unilamellarvesicles can be produced using methods known in the arts. e.g.,extrusion or sonication. Typically, small unilamellar vesicles areformed by sonication (e.g., tip or bath sonication) from largemultilamellar vesicles. Large unilamelar vesicles can be formed, forexample, by extrusion or by allowing small unilamellar vesicles tocoalesce.

Divalent cations are optionally present in the activation mixture. Insome aspects, divalent cations are present in the sample (e.g., arecalcified sample) and in the activation mixture. In other aspects,divalent cations can be added after the sample has contacted theactivation mixture.

In some aspects, the divalent cations are calcium ions. Any chemicalsource of calcium cations can be used, e.g., CaCl₂, Ca(NO₂)₂, CaSO₄, orother inorganic or organic calcium cation-containing compounds.

The methods disclosed herein can be applied to any sample containing acoagulation factor or suspected of containing a coagulation factor. Insome aspects, the sample can be whole blood, citrated or equivalentlystabilized blood, plasma, or other fluid sample containing or suspectedof containing a coagulation factor. In some aspects, the sample isdecalcified, e.g., decalcified plasma. Plasma can be decalcified, forexample, by adding chelators such as EDTA. In other aspects, the sampleis recalcified, e.g., recalcified plasma. Methods to decalcify bloodsamples, e.g., plasma, and specific conditions and calciumconcentrations for recalcification are well known in the art.

Measurements of coagulation, e.g., clotting time (Ct) measurements,using the activation mixtures disclosed herein, wherein the activationmixture is dried onto a solid substrate, can be carried out manually byvisual observation of clot formation. However, measurement ofcoagulation, e.g., clotting time (Ct) measurement, can also be performedusing optical or mechanical measurement instruments such as thosemarketed, e.g., by the Amelung, Baxter, Labor, Medtronic, CoaguSense,Roche Diagnostics (e.g., CoaguChek® I, II, XS; Coumatrak®),CardioVascular Diagnostics (e.g., TAS®), Organon Teknica (Coag-A-Mate®),Haemoscope (TEG), Pentapharm (ROTEM). Medirox, Siemens, Hemotek, HelenaLaboratories, and Behring companies. Measurements can also be performedusing point-of-care devised discussed infra.

The activation mixtures disclosed herein, wherein the activation mixtureis dried onto a solid substrate, can be applied to a variety of methodsfor measuring coagulation, and/or the concentration of coagulationfactors in biological samples, e.g., blood or plasma, and/or todetermine the effect or concentration of direct or indirect inhibitorsof coagulation. Such methods include both chromogenic assays andso-called “clotting methods” such as the aPTT assay. In general, these“clotting methods” are characterized by the fact that coagulation isactivated and the time from coagulation activation until detection ofclotting in the sample is measured, and in turn clotting time can beconverted into direct concentration units by establishing a calibrationcurve with appropriate calibration reagents.

In specific aspects of the present disclosure, the activation mixturecan be used as a reagent for the measurement of the Factor VIII activityof a Factor VIII protein (or a fragment, variant, derivative, chimericprotein, or hybrid protein thereof) in a sample. In one specific aspect,such activation mixture comprises 80% of 0.1 mg/mL Factor IXa and 20% ofa phospholipid mixture comprises 75 mole-% of phosphatidylcholine, 20mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol,wherein said activation mixture is dried onto a solid substrate. Inanother specific aspect, such activation mixture consist orsubstantially consists of 80% of 0.1 mg/mL Factor IXa and 20% of aphospholipid mixture comprises 75 mole-% of phosphatidylcholine, 20mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol,wherein said activation mixture is dried onto a solid substrate.

In specific aspects of the present disclosure, the activation mixturecan be used as a reagent for the measurement of the Factor IX activityof a Factor IX protein (or a fragment, variant, derivative, chimericprotein, or hybrid protein thereof) in a sample. In one specific aspect,such activation mixture comprises 80% of Factor XIa suspension and 20%of a phospholipid mixture comprising 75 mole-% of phosphatidylcholine,20 mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol,wherein said activation mixture is dried onto a solid substrate. Inanother specific aspect, such activation mixture consists orsubstantially consists of 80% of Factor XIa suspension and 20% of aphospholipid mixture comprising 75 mole-% of phosphatidylcholine, 20mole-% of phosphatidylserine, and 5 mole-% of phosphatidylglycerol,wherein said activation mixture is dried onto a solid substrate. Theexact amount of FXIa suspension needed varies depending on the specificactivity of this reagent and is titrated for optimal amount and mayinclude approximately 0.1 mg/mL to approximately 0.5 mg/mL.

Also provided in the present disclosure is a kit for performing ameasurement of coagulation factor activity or coagulation time in asample, wherein said kit comprises the components to prepare any of theactivation mixtures disclosed herein in one or more vials, as well asinstructions to dry the components to prepare any of the activationmixtures disclosed herein onto a solid substrate. Such kit can comprise,for example, (i) a solution comprising both an activated coagulationfactor and phospholipid vesicles in a single vial, or (ii) separatevials, one of them containing a solution of activated coagulation factorand a second vial containing a solution of phospholipid vesicles, or(ii) a vial containing a solution of activated coagulation factor and asecond vial containing a dried phospholipid mixture to be reconstitutedto produce phospholipid vesicles, etc. Thus, in some aspects, the kitcomprises one or more components in a dry form or non-dry form in one ormore vials, instructions for reconstituting or mixing the components inthe kit, and instruction for drying the activation mixture onto a solidsubstrate.

Also provided in the present disclosure is a sample holder forperforming a blood coagulation assay, comprising a surface coated withany one of the activation mixtures disclosed herein, wherein theactivation mixture is dried onto a solid substrate. For example, thesample holder can be a test strip, a test stick, a reaction chamber, acup, a cuvette, a cartridge, a chip, a well plate, an array, a membrane,a capillary, etc. A particular advantage of using a dried activationmixture coating a surface (as opposed to using a fluid reagent) in asample holder is that it extends the shelf life of the sample holder. Asecond advantage is that using a dried activation mixture applied tocoat the inner walls of a sample holder (e.g., a cartridge, a well or acuvette) is that the operator does not need to mix, pour, or otherwisedeal with liquid reactants.

In performing the assays disclosed herein (e.g., the Standard FMS assayor the Alternate FMS assay), a great variation in proteinconcentrations, incubation times, reagent concentrations, andtemperatures can be employed. The selection of particular assayparameters will depend on the coagulation factor to be assayed as wellas the source, type and size of the sample to be assayed, theanticipated levels of coagulation factor contained therein, and thethreshold of sensitivity desired. Taking these circumstances intoconsideration, selection of assay parameters will be apparent to thoseskilled in the art.

The assays disclosed herein (e.g., the Standard FMS assay or theAlternate FMS assay) can be used in methods for determining clottingtime in a patient having a bleeding disorder. Accordingly, the presentdisclosure provides a method for determining clotting time in a patienthaving bleeding disorder comprising (a) contacting a sample obtainedfrom the patient with an activation mixture comprising an activatedcoagulation factor and a phospholipid mixture, wherein the activationmixture is dried onto a solid substrate; and, (b) measuring the timebetween the contacting of the activation mixture with the blood sampleand the onset of clotting, thereby calculating the clotting time (Ct).

The present disclosure also provides a method of treating a patienthaving a bleeding disorder comprising (a) contacting a sample obtainedfrom the patient with an activation mixture comprising an activatedcoagulation factor and a phospholipid mixture, wherein the activationmixture is dried onto a solid substrate; (b) measuring the time betweenthe contacting of the activation mixture with the sample and the onsetof clotting, thereby calculating the clotting time (Ct), wherein Ctindicates whether the patient will benefit from administration of atreatment; and, (c) administering the treatment to the patient if Ctindicates that the patient will benefit from administration of thetreatment. Also provided is a method of treating a patient having ableeding disorder comprising (a) contacting a sample obtained from thepatient with an activation mixture comprising an activated coagulationfactor and a phospholipid mixture, wherein the activation mixture isdried onto a solid substrate; (b) measuring the time between thecontacting of the activation mixture with the sample and the onset ofclotting, thereby calculating the clotting time (Ct), wherein Ctindicates whether the patient will benefit from administration of atreatment; and, (c) instructing a healthcare provider to administer thetreatment to the patient if Ct indicates that the patient will benefitfrom administration of the treatment.

The disclosure also provides a method of optimizing a bleeding disordertreatment in a patient comprising (a) contacting a sample obtained fromthe patient with an activation mixture comprising an activatedcoagulation factor and a phospholipid mixture, wherein the activationmixture is dried onto a solid substrate; (b) measuring the time betweenthe contacting of the activation mixture with the sample and the onsetof clotting, thereby calculating the clotting time (Ct), wherein Ctcorrelates with a therapeutically efficacious treatment; and, (c)administering an optimized treatment to the patient, wherein thetreatment is maintained or adjusted. Also provided is a method ofoptimizing a bleeding disorder treatment in a patient comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct), wherein Ct correlates with a therapeutically efficacioustreatment; and, (c) instructing a healthcare provider to optimize thetreatment administered, wherein the treatment is maintained or adjusted.

The instant disclosure also provides a method of diagnosing whether apatient is in need of treatment for a bleeding disorder comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct), wherein Ct indicates whether the patient has a bleedingdisorder; and, (c) providing a treatment for the bleeding disorder ifthe patient is in need thereof. Also provided is a method of diagnosingwhether a patient is in need of treatment for a bleeding disordercomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct), wherein Ct indicates whether thepatient has a bleeding disorder; and, (c) instructing a healthcareprovider to provide treatment for the bleeding disorder if the patientis in need thereof.

Also provided in the present disclosure is a method of monitoring theefficacy of a bleeding disorder treatment administered to a patientcomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); and, (c) comparing the measured Ctwith the Ct obtained from a corresponding standard, wherein the standardis representative of a therapeutically efficacious treatment, andwherein a similarity between the patient's results and the standard isindicative of efficacy of the patient's current treatment; and, (d)maintaining or adjusting the patient's treatment based on the relativedifference between the patient's results and the corresponding standard.Also provided is a method of monitoring the efficacy of a bleedingdisorder treatment administered to a patient comprising (a) contacting asample obtained from the patient with an activation mixture comprisingan activated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct);and, (c) comparing the measured Ct with the Ct obtained from acorresponding standard, wherein the standard is representative of atherapeutically efficacious treatment, and wherein a similarity betweenthe patient's results and the standard is indicative of efficacy of thepatient's current treatment; and, (d) instructing a healthcare providerto maintain or adjusting the patient's treatment based on the relativedifference between the patient's results and the corresponding standard.

The present disclosure also provides a method for determining acoagulation factor level in a bleeding disorder patient, comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct); and, (c) correlating the Ct value with the level ofcoagulation factor in the sample. The correlation between Ct andcoagulation factor level (% Factor) can be calculated, for example,according to the formula:Ct=A×Ln(% Factor)+Bwherein, for each coagulation factor, A is a constant valuecorresponding to the slope of a Ct versus coagulation factorconcentration dose-response, and B is patient-specific off-set value.

For a given coagulation factor, the A values for dose response curvesplotting concentration of coagulation factor (% Factor) versus Ct aresimilar for all patients, whereas the B off-set values are different dueto patient-specific global coagulation differences. The variability in Bvalues can be addressed, for example, by optimizing the chemistry of theactivation mixture so that there is no difference in B values amongpatients. The resulting correlation between concentration of factor andCt can be used in a “Ready to Use Factor Monitoring Device” that doesnot require patient-specific calibration. Such device can be, forexample, a point-of-care device.

Alternatively, the variability in B values can be addressed bycustomizing the device for each patient. For example, Ct can be measuredduring an initial (training) visit using the Standard FMS assay orAlternate FMS assay disclosed herein, and venous sample for standardlaboratory analysis can be obtained at the same time. The BI value,offset between Ct value from FMS assay(s) and the laboratory assays,could be provided to the patient (e.g., as an ID value). The ID valuecould be used to program the device, thus providing a “Customized FactorMonitoring Device” specifically customized for a single patient.Multiple patient IDs would be possible per device. Such device can be,for example, a point-of-care device.

The instant disclosure also provides a method for determining apharmacokinetic (PK) parameter in a bleeding disorder patient,comprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); and, (c) correlating a PK with thecalculated Ct value, thereby determining the value of the PK parameter.

Also provided in the present disclosure is a method of treating apatient having a bleeding disorder comprising (a) contacting a sampleobtained from the patient with an activation mixture comprising anactivated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct);(c) determining a PK parameter based on Ct, wherein the PK parameterindicates that the patient will benefit from administration of thetreatment; and, (d) administering the treatment to the patient if the PKparameter indicates that the patient will benefit from administration ofthe treatment. Also provided in the present disclosure is a method oftreating a patient having a bleeding disorder comprising (a) contactinga sample obtained from the patient with an activation mixture comprisingan activated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct);(c) determining a PK parameter based on Ct, wherein the PK parameterindicates that the patient will benefit from administration of thetreatment; and, (d) instructing a healthcare provider to administer thetreatment to the patient if the PK parameter indicates that the patientwill benefit from administration of the treatment.

The present disclosure also provides is a method of optimizing ableeding disorder treatment in a patient comprising (a) contacting asample obtained from the patient with an activation mixture comprisingan activated coagulation factor and a phospholipid mixture, wherein theactivation mixture is dried onto a solid substrate; (b) measuring thetime between the contacting of the activation mixture with the sampleand the onset of clotting, thereby calculating the clotting time (Ct);(c) determining a PK parameter based on Ct, wherein the PK parametercorrelates with a therapeutically efficacious treatment; and, (d)administering an optimized treatment to the patient, wherein thetreatment is maintained or adjusted. The present disclosure alsoprovides a method of optimizing a bleeding disorder treatment in apatient comprising (a) contacting a sample obtained from the patientwith an activation mixture comprising an activated coagulation factorand a phospholipid mixture, wherein the activation mixture is dried ontoa solid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); (c) determining a PK parameter basedon Ct, wherein the PK parameter correlates with a therapeuticallyefficacious treatment; and, (d) instructing a healthcare provider toadminister an optimized treatment to the patient, wherein the therapy ismaintained or adjusted.

The instant disclosure also provides a method of diagnosing whether apatient is in need of treatment for a bleeding disorder comprising (a)contacting a sample obtained from the patient with an activation mixturecomprising an activated coagulation factor and a phospholipid mixture,wherein the activation mixture is dried onto a solid substrate; (b)measuring the time between the contacting of the activation mixture withthe sample and the onset of clotting, thereby calculating the clottingtime (Ct); (c) determining a PK parameter based on Ct, wherein the PKparameter indicates whether the patient has a bleeding disorder; and,(d) providing treatment for the bleeding disorder if the patient is inneed thereof.

Also provided in the instant disclosure is a method of diagnosingwhether a patient is in need of treatment for a bleeding disordercomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); and, (c) determining a PK parameterbased on Ct, wherein the PK parameter indicates whether the patient hasa bleeding disorder; and, (d) instructing a healthcare provider toprovide therapy to treat the bleeding disorder if the patient is in needthereof.

The present disclosure also provides a method of monitoring the efficacyof a bleeding disorder treatment administered to a patient comprising(a) contacting a sample obtained from the patient with an activationmixture comprising an activated coagulation factor and a phospholipidmixture, wherein the activation mixture is dried onto a solid substrate;(b) measuring the time between the contacting of the activation mixturewith the sample and the onset of clotting, thereby calculating theclotting time (Ct); and, (c) determining a PK parameter based on Ct; (d)comparing the PK parameter with the PK obtained from a correspondingstandard, wherein the standard is representative of a therapeuticallyefficacious treatment, and wherein a similarity between the patient'sresults and the standard is indicative of efficacy of the patient'scurrent treatment; and, (e) maintaining or adjusting the patient'streatment based on the relative difference between the patient's resultsand the corresponding standard. Also provided is a method of monitoringthe efficacy of a bleeding disorder treatment administered to a patientcomprising (a) contacting a sample obtained from the patient with anactivation mixture comprising an activated coagulation factor and aphospholipid mixture, wherein the activation mixture is dried onto asolid substrate; (b) measuring the time between the contacting of theactivation mixture with the sample and the onset of clotting, therebycalculating the clotting time (Ct); (c) determining a PK parameter basedon Ct; (d) comparing the PK parameter with the PK obtained from acorresponding standard, wherein the standard is representative of atherapeutically efficacious treatment, and wherein a similarity betweenthe patient's results and the standard is indicative of efficacy of thepatient's current treatment; and, (e) instructing a healthcare providerto maintain or adjust the patient's treatment based on the relativedifference between the patient's results and the corresponding standard.

In some aspects, the PK is terminal half-life (“HL”). In other aspects,the PK is time to through (“T”). The PK parameters disclosed herein aswell as other PK parameters known in the art can be calculated from Ctand additional parameters that can be determined experimentally and/orfrom pharmacodynamic simulation and/or pharmacokinetic simulations. Forexample, pharmacokinetic and pharmacodynamics parameters can becalculated for a certain coagulation factor, for a certain population,or for a certain administration route, dosage, or other condition basedon simulations conducts on data obtained from a single patient or frommultiple patients (e.g., patients in a clinical trial).

In some aspects, HL can be calculated according to the formula:HL=−0.693×(T ₂ −T ₁)×A/(Ct₁−Ct₂)

wherein, for each coagulation factor. A is a constant valuecorresponding to the slope of a Ct versus coagulation factorconcentration dose-response, T₁ and T₂ are times at which Ct ismeasured, and Ct₁ and Ct₂ are Ct values measured at T₁ and T₂,respectively. In this calculation, the offset value B becomesirrelevant. i.e., interpatient differences in global coagulation do notaffect terminal half-life. The possibility of repeating Ct measures on apoint-of-care device on multiple days applying the method andcompositions disclosed herein (for example, one measurement per day for5 to 8 days) means that the likely result would be far more accuratethan terminal half-life values obtained using one or two traditionallaboratory-based measurements.

In some aspects, the patient-specific terminal half-life calculatedaccording to the method disclosed above can be combined withpharmacokinetic and/or pharmacodynamics data. For example,product-specific in vivo recovery and distribution phase (α-phase)half-life data can be obtained via population modeling using dataobtained from clinical trials. “In vivo recovery” (“IVR”) is generallyrepresented by the incremental recovery (K-value), which is the observedpeak activity minus predose level and then divided by the dose. IVR canalso be calculated on a percentage basis. The mean IVR can be determinedin a patient population, or the individual IVR can be determined in asingle subject. Product-specific in vivo recovery and distribution phase(α-phase) half-life data can be combined to patient-specific terminalhalf-life data to calculate time to trough (T) according to the formula:T=−1.44×HL/(A×(Ct_(measured)−Ct_(trough))wherein for each coagulation factor A is a constant value correspondingto the slope of a Ct versus coagulation factor concentrationdose-response, and HL is the terminal half-life, Ct_(measured) is Ctmeasured at certain time point, and Ct_(trough) is patient-specific clottime at trough. In some aspects, the patient is administered a new doseof coagulation factor every T interval.

In some aspects, the sample used in the methods of treating, optimizinga treatment, diagnosing whether a patient needs a treatment, monitoringthe efficacy of the treatment, or in the methods for determiningclotting times, coagulation factor levels, and pharmacokinetic (PK)parameters disclosed herein, comprises, e.g., whole blood, citrated orequivalently stabilized blood, plasma, or other fluid sample containingor suspected of containing a coagulation factor. In some aspects, thesample is whole blood, for example venous blood obtained via phlebotomy,whereas in other aspects the blood is fingerstick blood. In somespecific aspects, a single drop of fingerstick blood is required topractice the disclosed methods.

In other aspects, the sample is plasma. Samples, e.g., plasma or blood,can be refrigerated or used at room temperature. In some aspects,samples, e.g., plasma or blood, can be frozen and thawed prior tocontacting the sample with the activation mixture. In other cases, thesample has not been frozen and thawed prior to contacting the samplewith the activation mixture. In some aspects, the sample is decalcified,e.g., by adding a chelator such as EDTA to the sample. In other aspects,the decalcified sample is recalcified prior to contacting the samplewith the activation mixture by adding a solution containing divalentions, e.g., calcium ions. In certain aspects, the decalcified sample isrecalcified after contacting the sample with the activation mixture.

In certain aspects, variability between samples can be reduced byadding, for example, a purified coagulation factor or an inhibitor ofcoagulation to the sample. Purified coagulation factor that can be addedto the sample include, for example, Factors II, Factor VII, Factor VIII,Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, Fibrinogen,vWF, Tissue Factor, and combinations thereof. Coagulation inhibitorsthat can be added to the sample include, for example, CTI, aprotinin,ε-aminocaproic acid (EACA). D-Phenylalanyl-1-prolyl-1-argininechloromethyl ketone-Factor VIIa (FPRCK-FVIIa), anti-coagulation factormonoclonal antibodies, and combinations thereof. In some aspects, theone purified coagulation factor can be added to the sample. In othercases, more than one purified coagulation factor can be added to thesample. In some cases, one coagulation inhibitor can be added to thesample. In other cases, more than one coagulation inhibitor can be addedto the sample. In some cases, a combination comprising at least onepurified coagulation factor and at least one coagulation inhibitor canbe added to the sample.

In some cases, the sample can be diluted, for example, with substratesample (i.e., sample that has been depleted of the assay target factor).This dilution can consist, for example, of one part of sample dilutedwith three parts of substrate sample. In some aspects, the sample isdiluted with substrate sample at about a 1:2 ratio, at about 1:3 ratio,at about a 1:4 ratio, or at about a 1:5 ratio. Dilution ratios can beadjusted above or below the disclosed ratios using routineexperimentation.

In some aspects, the methods of treating, optimizing a treatment,diagnosing whether a patient needs a treatment, monitoring the efficacyof the treatment, or in the methods for determining clotting times,coagulation factor levels, and pharmacokinetic (PK) parameters disclosedherein, use an activation mixture comprising an activated coagulationfactor wherein the factor is a Factor IXa protein or a fragment,variant, or derivative thereof.

In some aspects, the methods of treating, optimizing a treatment,diagnosing whether a patient needs a treatment, monitoring the efficacyof the treatment, or in the methods for determining clotting times,coagulation factor levels, and pharmacokinetic (PK) parameters disclosedherein, use an activation mixture comprising an activated coagulationfactor wherein the factor is a Factor XIa protein or a fragment,variant, or derivative thereof.

In some aspects, the methods of treating, optimizing a treatment,diagnosing whether a patient needs a treatment, monitoring the efficacyof the treatment, or in the methods for determining clotting times,coagulation factor levels, and pharmacokinetic (PK) parameters disclosedherein, use an activation mixture comprising a phospholipid mixture.This phospholipid mixture can comprise, for example, 1 phospholipid, 2phospholipids, 3 phospholipids, or more than 3 phospholipids. Thesephospholipids can be, for example, phosphatidylcholine,phosphatidylserine, or phosphatidylglycerol. The phospholipids in thephospholipid mixture can be, for example, natural phospholipids,synthetic phospholipids, or combinations thereof.

In some specific aspects, the phospholipid mixture comprises 70 mole-%of phosphatidylcholine and 30 mole-% of phosphatidylserine. In certainspecific aspects, the phospholipid mixture consists or essentiallyconsists of 70 mole-% of phosphatidylcholine and 30 mole-% ofphosphatidylserine. In other aspects, the phospholipid mixture comprises80 mole-% of phosphatidylcholine, 10 mole-% of phosphatidylserine, and10 mole-% of phosphatidylglycerol. In yet other aspects, thephospholipid mixture consists or essentially consists of 80 mole-% ofphosphatidylcholine, 10 mole-% of phosphatidylserine, and 10 mole-% ofphosphatidylglycerol. In some aspects, the phospholipid mixturecomprises 75 mole-% of phosphatidylcholine, 20 mole-% ofphosphatidylserine, and 5 mole-% of phosphatidylglycerol. In otheraspects, the phospholipid mixture consists or essentially consists of 75mole-% of phosphatidylcholine, 20 mole-% of phosphatidylserine, and 5mole-% of phosphatidylglycerol. In certain aspects, the phospholipidmixture further comprises cholesterol, for example at a concentrationfrom about 1 to about 20 mole-% of cholesterol.

In some aspects, the activation mixture used in the methods of treating,optimizing a treatment, diagnosing whether a patient needs a treatment,monitoring the efficacy of the treatment, or in the methods fordetermining clotting times, coagulation factor levels, andpharmacokinetic (PK) parameters disclosed herein, comprises aphospholipid mixture in lipid vesicle form. In some aspects, the lipidvesicles are small unilamellar vesicles.

In some aspects, the activation mixture used in the methods of treating,optimizing a treatment, diagnosing whether a patient needs a treatment,monitoring the efficacy of the treatment, or in the methods fordetermining clotting times, coagulation factor levels, andpharmacokinetic (PK) parameters disclosed herein, further comprisesdivalent cations. e.g., calcium ions.

In some aspects, the activation mixture used in the methods of treating,optimizing a treatment, diagnosing whether a patient needs a treatment,monitoring the efficacy of the treatment, or in the methods fordetermining clotting times, coagulation factor levels, andpharmacokinetic (PK) parameters disclosed herein, can react with acoagulation factor, e.g., Factor VII. Factor VIII, or Factor IX. In someaspects, the Factor VIII coagulation factor is a Factor VIII protein (ora fragment, variant, derivative, chimeric protein, or hybrid proteinthereof). In some aspects, the Factor VIII coagulation factor is achimeric Factor VIII-Fc fusion protein. In some aspects, the Fc portionof the chimeric Factor VIII protein comprises a human Fc domain. In someaspects, the chimeric Factor VIII protein comprises a B-domain deletedFactor VIII. In specific aspects, the chimeric Factor VIII proteincomprises SEQ ID NO:6, or SEQ ID NO:2.

In other aspects, the Factor IX coagulation factor is a Factor IXprotein (or a fragment, variant, derivative, chimeric protein, or hybridprotein thereof). In some aspects, the Factor IX coagulation factor is achimeric Factor IX-Fc fusion protein. In some aspects, the Fc portion ofthe chimeric Factor IX protein comprises a human Fc domain. In certainspecific aspects, the chimeric Factor IX protein comprises SEQ ID NO:13.

In some aspects, the activation mixture used in the methods of treating,optimizing a treatment, diagnosing whether a patient needs a treatment,monitoring the efficacy of the treatment, or in the methods fordetermining clotting times, coagulation factor levels, andpharmacokinetic (PK) parameters disclosed herein, is dried onto a thesolid substrate. This solid substrate can be, for example, paper,plastic, glass, ceramic material, metal, and combinations thereof. Insome aspects, the solid substrate is a surface on a test strip, teststick, reaction chamber, cartridge, chip, well plate, or array used inan apparatus to measure coagulation factor activity or coagulation time.

In some aspects, the patient in the methods of treating, optimizing atreatment, diagnosing whether a patient needs a treatment, monitoringthe efficacy of the treatment, or in the methods for determiningclotting times, coagulation factor levels, and pharmacokinetic (PK)parameters disclosed herein, has not yet been treated with a coagulationfactor. However, in other cases, the patient has received priorcoagulation factor treatment, but the treatment has been discontinuedfor a time period sufficient to deplete the coagulation factor treatmentfrom the patient's blood.

The methods, compositions, and systems of the present disclosure can beapplied to treating a patient or evaluating or determining whether apatient will benefit from administration of a therapeutically effectivedose of a therapeutic agent that is capable of treating a bleedingdisorder, for example, hemophilia A or hemophilia B. The methods ofsystems disclosed herein can be used to apply more precise coagulationfactor dosing to patients. In a further aspect, the methods and systemsdisclosed herein can be used to increase the power and effectiveness ofclinical trials. Thus, individuals in a study can be monitored anddosages adjusted individually. When the methods of the presentdisclosure are used for the treatment of bleeding disorders byadministration of a coagulation factor. e.g., a Factor VIII or Factor IXprotein (or fragment, variants, derivative, chimeric proteins, or hybridprotein thereof), individualized treatment using the methods providedherein can result in fewer disease flare-ups, and thus provide a higherquality of life for the patient. In order to treat a patient, samplesfrom the patient can be obtained before or after the administration of aFVIII or FIX polypeptide. In some cases, successive samples can beobtained from the patient after clotting factor treatment has commencedor after treatment has ceased.

Samples can, e.g., be requested by a healthcare provider (e.g., adoctor) or healthcare benefits provider, obtained and/or processed bythe same or a different healthcare provider (e.g., a nurse, a hospital)or a clinical laboratory, and after processing, the results can beforwarded to yet another healthcare provider, healthcare benefitsprovider or the patient. Similarly, the measuring/determination ofclotting times, the comparisons between time points, and treatmentdecisions can be performed by one or more healthcare providers,healthcare benefits providers, and/or clinical laboratories. In somecases, the methods, compositions, and systems disclosed herein can beapplied in a point-of-care test system.

The methods described herein can be used for variety of evaluations,including without limitation, analysis of a patient's blood prior totreatment (or after complete washout of prior therapeutic treatment, toevaluate ‘baseline’ clot formation (which can correlate with severity ofthe disease) and adding various therapeutic composition(s) such asrecombinant FVIII or FIX ex vivo to such blood in order to predict theindividual's response to therapy. The methods disclosed herein can beapplied, for example, to measure clotting time in samples from a patientsuffering from a bleeding disorder, samples from a patient sufferingfrom a clotting disorder, or samples from a healthy patient (e.g., priorto surgery). The methods disclosed herein can also be applied, forexample, to determine the effect on coagulation of a natural,recombinant, or chimeric clotting factor, a biological (e.g., anantibody or fusion protein), an anticoagulant, or a small molecule drugadded to a plasma or blood sample, or present or suspected to be presentin a blood or plasma sample from a patient. Thus, the methods disclosedherein are generally applicable to the measurement of coagulation (e.g.,by measuring clotting time) in samples from patients suffering or atrisk of suffering conditions other bleeding disorders other thanhemophilias. For example, in some conditions such as lupus, coagulationcan be altered by the presence of lupus anticoagulant, a prothromboticagent that precipitates the formation of thrombi in vivo. Patients withlupus and other conditions causing thrombosis can be treated withanticoagulants. Coagulation can also be altered by substances fromanimal origin, e.g., hirudin or proteins from snake venoms. Certain drugtherapies, for example, warfarin treatment, are known to influencecoagulation factor levels. Also patients suffering from consumptivecoagulapathies such as thrombosis or disseminated intravascularcoagulation (DIC) can present anomalies in coagulation factor levelswhich require careful clinical management. Successful treatment of theseconditions similarly requires accurate determination of serumcoagulation factor levels. In managing any of the aforementioned medicalconditions, one mode of treatment involves administration of exogenouscoagulation factors (e.g., Factor VIII or FIX proteins, fragments,variants or derivatives, for example rFVIIIFc or rFIXFc). It isessential that the precise concentration of such therapeutic doses bemeasured, and the quantity of coagulation factor be monitored.

Accordingly, the methods for diagnosing, treating, optimizing treatment,monitoring treatment, etc. disclosed herein can generally be applied toany diseases, conditions, or any situations in which blood coagulationis compromised or is suspected to be compromised, and also forprophylactic or preventive purposes (for example, to detect the onset ofa disease or condition in a patient at risk or with a family history ofsuch disease).

III. Point-of-Care Applications

In many situations, blood coagulation tests can be performed directly atthe point of care without transport of the sample to an separatefacility. e.g., a laboratory. The advantages of point-of-care analysisinclude (i) short turn-around time, as there is no time or only littletime needed for transport of the sample, which allows fastmonitoring-directed decisions, (ii) transport of the sample to anemergency laboratory can be very expensive, especially at night and whenonly few samples are to be analyzed, and (iii) self-testing of thepatient is possible.

Available point-of-care methods methods for analysis of coagulation timehave the same limitations as the determination of aPTT in thelaboratory, e.g., non-linear dose response, low sensitivity, or highvariation between samples and/or patients.

The methods and compositions of the present disclosure can be used inimproved assays for point-of-care analysis of samples. e.g., bloodsamples such as whole blood samples. Thus, the present disclosure alsoincludes a point-of-care hematological assay wherein the activationmixture disclosed herein is positioned in one or more reaction locationsin a test apparatus and a sample of body fluid to be assayed (e.g.,whole blood, citrated blood, or plasma) is contacted by the activationmixture.

As a specific aspect, the present disclosure provides a point-of-caredevice designed to rapidly test for coagulation levels, e.g., levels ofcoagulation factor VIII (FVIII) or factor IX (FIX) levels in hemophiliapatients, from a finger stick blood sample by using raw clotting times,wherein said point of care device uses a disposable sample holder (e.g.,a disposable strip) coated with a activation mixture comprising anactivated coagulation factor and a phospholipid mixture, and whereinsaid activation mixture is dried onto said disposable sample holder.

The Standard FMS and Alternate FMS assays disclosed herein can beimplemented in point-of-care devices and used as a global hemostasistests by using raw clotting times (Ct) to determine individualpharmacokinetic parameters which in term can be used to decidetreatment. Accordingly, the methods and composition disclosed herein canbe applied to measure coagulation activity by implementing them incommercially available point-of-care self-monitoring devices, forexample, i-STAT 1 (Abbott Point of Care); INRatio or INRatio2 PT INRMonitors (Alere); RapidPoint (Bayer); Coag-Sense PT/INR MonitoringSystem (CoaguSense); Actalyke Mini II, Actalyke XL, or Cascade POC(Helena Point of Care); Gem PCL Plus (Instrumentation Laboratory);Hemochrom Response, Hemochron Signature Elite, Hemochron Signature+, orProTime Microcoagulation System (ITC); ACT Plus, or HMS Plus (MedtronicCardiac Surgery); CoaguCheck XS Pro PT, CoaguCheck XS PT, CoaguCheckPlus PT (Roche Diagnostics); etc.

IV. Factor VIII and Factor IX Polypeptides

The methods and compositions provided in the present disclosure can beused in assays to determine the activity of clotting factors, such asFactor VIII and Factor IX polypeptides (including fragments, variants,derivatives, chimeric, and hybrid polypeptides). A detailed descriptionof Factor VIII and Factor IX polypeptides (including fragments,variants, derivatives, chimeric and hybrid polypeptides) whosecoagulating activity can be assessed by using the methods andcompositions of the present disclosure is provided below.

A. Factor VIII Polypeptides

“Factor VIII.” as used herein, means functional Factor VIII polypeptidein its normal role in coagulation, unless otherwise specified. Thus, theterm Factor VIII includes variant polypeptides that are functional.Factor VII proteins include the human, porcine, canine, and murineFactor VIII proteins. The full length polypeptide and polynucleotidesequences are known, as are many functional fragments, mutants andmodified versions. Examples of human Factor VIII sequences are shown assubsequences in SEQ ID NOs: 2, 6, 8, 10, and 12 (Sequence Table 2).Factor VIII polypeptides include, e.g., full-length Factor VIII,full-length Factor VIII minus Met at the N-terminus, mature Factor VIII(minus the signal sequence), mature Factor VIII with an additional Metat the N-terminus, and/or Factor VIII with a full or partial deletion ofthe B domain. Factor VIII polypeptides include B domain deletions,whether partial or full deletions or single chain FVIII. Factor VIII canbe made by recombinant means (“recombinant Factor VIII” or “rFVIII”),i.e., it is not naturally occurring or derived from plasma.

“B domain” of Factor VIII, as used herein, is the same as the B domainknown in the art that is defined by internal amino acid sequenceidentity and sites of proteolytic cleavage by thrombin, e.g., residuesSer741-Arg1648 of full length human Factor VIII. The other human FactorVIII domains are defined by the following amino acid residues: A1,residues Ala1-Arg372; A2, residues Ser373-Arg740; A3, residuesSer1690-Ile2032; C1, residues Arg2033-Asn2172; C2, residuesSer2173-Tyr2332. The A3-C1-C2 sequence includes residuesSer1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, isusually referred to as the Factor VIII light chain activation peptide.The locations of the boundaries for all of the domains, including the Bdomains, for porcine, mouse and canine Factor VIII are also known in theart. In certain aspects, the B domain of Factor VIII is deleted (“Bdomain deleted Factor VIII” or “BDD FVIII”). An example of a BDD FVIIIis REFACTO (recombinant BDD FVIII), which has the same sequence as theFactor VIII portion of the sequence in Sequence Table 2A(i) (amino acids−19 to 1438 or 1 to 1438 of SEQ ID NO:2).

A “B domain deleted Factor VIII” can have the full or partial deletionsdisclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203,6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502,5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of whichis incorporated herein by reference in its entirety. In some aspects, aB domain deleted Factor VIII sequence of the present disclosurecomprises any one of the deletions disclosed at col. 4, line 4 to col.5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S.Pat. No. 6,346,513). In some aspects, a B domain deleted Factor VIII ofthe present disclosure has a deletion disclosed at col. 2, lines 26-51and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. No.6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620). Insome aspects, a B domain deleted Factor VIII has a deletion described incol. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6,lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S.Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 of U.S.Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 ofU.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11,line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines25-53, of U.S. Pat. No. 6,458,563. In some aspects, a B domain deletedFactor VIII has a deletion of most of the B domain, but still containsamino-terminal sequences of the B domain that are essential for in vivoproteolytic processing of the primary translation product into twopolypeptide chain, as disclosed in WO 91/09122, which is incorporatedherein by reference in its entirety. In some aspects, a B domain deletedfactor VIII is constructed with a deletion of amino acids 747-1638.e.g., virtually a complete deletion of the B domain. Hoeben R. C., etal. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein byreference in its entirety. A B domain deleted Factor VIII can alsocontain a deletion of amino acids 771-1666 or amino acids 868-1562 offactor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988),incorporated herein by reference in its entirety. Additional B domaindeletions include, e.g.: deletion of amino acids 982 through 1562 or 760through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83,5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986)25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No.WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741through 1648 (Pasek (PCT application No. 88/00831)), 816 through 1598 or741 through 1689 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP295597)), each of which is incorporated herein by reference in itsentirety.

In other aspects, BDD FVIII includes a FVIII polypeptide containingfragments of the B-domain that retain one or more N-linked glycosylationsites, e.g., residues 757, 784, 828, 900, 963, or optimally 943, whichcorrespond to the amino acid sequence of the full-length FVIII sequence.Examples of the B-domain fragments include 226 amino acids or 163 aminoacids of the B-domain as disclosed in Miao, H. Z., et al., Blood 103(a):3412-3419 (2004). Kasuda, A, et al., J. Thromb. Haemost. 6: 1352-1359(2008), and Pipe, S. W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011)(e.g., the first 226 amino acids or 163 amino acids of the B domain areretained). In still other aspects, BDD FVIII further comprises a pointmutation at residue 309 (from Phe to Ser) to improve expression of theBDD FVIII protein. See Miao. H. Z., et al., Blood 103(a): 3412-3419(2004). In still other aspects, the BDD FVIII includes a FVIIIpolypeptide containing a portion of the B-domain, but not containing oneor more furin cleavage sites (e.g., Arg1313 and Arg 1648). See Pipe, S.W., et al., J. Thromb. Haemost. 9: 2235-2242 (2011). The references areincorporated herein by reference, and each of the foregoing deletionscan be made in any Factor VIII sequence.

In certain aspects, FVIII includes a single chain FVIII polypeptide. Inone embodiment, a single chain FVIII polypeptide can include one or moremutations or substitutions at R1645 or R1648 corresponding tofull-length Factor VIII sequence or both. Additional examples of singlechain FVIII polypeptides can be found at U.S. Provisional ApplicationNo. 61/668,889, filed Jul. 6, 2012, which is incorporated herein byreference in its entirety. In another embodiment, a single chain FVIIIpolypeptide contains a FVIII polypeptide having a deletion of R1645and/or R1648 corresponding to full-length FVIII sequence or a sequencecontaining R1645 and/or R1648 corresponding to full-length FVIII. Forexample, a single chain FVIII can contain a deletion of amino acidpositions 746 to 1649, 746 to 1652, 746 to 1655, 758 to 1649, 758 to1652, 758 to 1655, 765 to 1649, 765 to 1652, 765 to 1655, 748 to 1658,755 to 1658, 762 to 1658, 769 to 1658, 776 to 1658, or 783 to 1658corresponding to full-length FVIII sequence. Additional examples can befound at U.S. Pat. No. 7,041,635, filed Jan. 3, 2003, which isincorporated herein by reference in its entirety.

A great many functional Factor VIII variants are known, as is discussedabove and below. In addition, hundreds of nonfunctional mutations inFactor VIII have been identified in hemophilia patients, and it has beendetermined that the effect of these mutations on Factor VIII function isdue more to where they lie within the 3-dimensional structure of FactorVIII than on the nature of the substitution (Cutler et al., Hum. Mutat.19:274-8 (2002), incorporated herein by reference in its entirety). Inaddition, comparisons between Factor VIII from humans and other specieshave identified conserved residues that are likely to be required forfunction (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat.No. 6,251,632), incorporated herein by reference in its entirety.

The human Factor VIII gene was isolated and expressed in mammalian cells(Toole, J. J., et al. Nature 312:342-347 (1984); Gitschier. J., et al.,Nature 312:326-330 (1984); Wood, W. I., et al., Nature 312:330-337(1984); Vehar. G. A., et al., Nature 312:337-342 (1984); WO 87/04187; WO88/08035; WO 88/03558; U.S. Pat. No. 4,757,006), each of which isincorporated herein by reference in its entirety, and the amino acidsequence was deduced from cDNA. Capon et al., U.S. Pat. No. 4,965,199,incorporated herein by reference in its entirety, discloses arecombinant DNA method for producing Factor VIII in mammalian host cellsand purification of human Factor VIII. Human Factor VIII expression inCHO (Chinese hamster ovary) cells and BHKC (baby hamster kidney cells)has been reported. Human Factor VIII has been modified to delete part orall of the B domain (U.S. Pat. Nos. 4,994,371 and 4,868,112, each ofwhich is incorporated herein by reference in its entirety), andreplacement of the human Factor VIII B domain with the human Factor V Bdomain has been performed (U.S. Pat. No. 5,004,803, incorporated hereinby reference in its entirety). The cDNA sequence encoding human FactorVIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2,respectively, of US Application Publ. No. 2005/0100990, incorporatedherein by reference in its entirety.

U.S. Pat. No. 5,859,204. Lollar. J. S., incorporated herein by referencein its entirety, reports functional mutants of Factor VIII havingreduced antigenicity and reduced immunoreactivity. U.S. Pat. No.6,376,463, Lollar. J. S., incorporated herein by reference in itsentirety, also reports mutants of Factor VIII having reducedimmunoreactivity. US Application Publ. No. 2005/0100990, Saenko et al.,incorporated herein by reference in its entirety, reports functionalmutations in the A2 domain of Factor VIII.

A number of functional Factor VIII molecules, including B-domaindeletions, are disclosed in the following patents U.S. Pat. No.6,316,226 and U.S. Pat. No. 6,346,513, both assigned to Baxter; U.S.Pat. No. 7,041,635 assigned to In2Gen; U.S. Pat. No. 5,789,203, U.S.Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620assigned to Chiron; U.S. Pat. No. 5,972,885 and U.S. Pat. No. 6,048,720assigned to Biovitrum, U.S. Pat. No. 5,543,502 and U.S. Pat. No.5,610,278 assigned to Novo Nordisk; U.S. Pat. No. 5,171,844 assigned toImmuno Ag; U.S. Pat. No. 5,112,950 assigned to Transgene S.A.; U.S. Pat.No. 4,868,112 assigned to Genetics Institute, each of which isincorporated herein by reference in its entirety.

The porcine Factor VIII sequence is published, (Toole, J. J., et al.,Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986)), incorporated herein byreference in its entirety, and the complete porcine cDNA sequenceobtained from PCR amplification of factor VIII sequences from a pigspleen cDNA library has been reported (Healey, J. F., et al., Blood88:4209-4214 (1996), incorporated herein by reference in its entirety).Hybrid human/porcine Factor VIII having substitutions of all domains,all subunits, and specific amino acid sequences were disclosed in U.S.Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093, incorporatedherein by reference in its entirety. More recently, the nucleotide andcorresponding amino acid sequences of the A1 and A2 domains of porcinefactor VIII and a chimeric Factor VIII with porcine A1 and/or A2 domainssubstituted for the corresponding human domains were reported in WO94/11503, incorporated herein by reference in its entirety. U.S. Pat.No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA anddeduced amino acid sequences. 6,458,563, incorporated herein byreference in its entirety assigned to Emory discloses a B-domain deletedporcine Factor VIII.

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) canbe at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Sequence Table 2 without a signal sequence (amino acids 1 to1438 of SEQ ID NO:2; amino acids 1 to 2332 of SEQ ID NO:6; amino acids 1to 740 of SEQ ID NO:8; amino acids 1 to 745 of SEQ ID NO:10; or aminoacids 1 to 684 of SEQ ID NO:12). The Factor VIII (or Factor VIII portionof a chimeric polypeptide) can be identical to a Factor VIII amino acidsequence shown in Sequence Table 2 without a signal sequence (aminoacids 1 to 1438 of SEQ ID NO:2; amino acids 1 to 2332 of SEQ ID NO:6;amino acids 1 to 740 of SEQ ID NO:8; amino acids 1 to 745 of SEQ IDNO:10; or amino acids 1 to 684 of SEQ ID NO: 12).

The Factor VIII (or Factor VIII portion of a chimeric polypeptide) canbe at least 90% or 95% identical to a Factor VIII amino acid sequenceshown in Sequence Table 2 with a signal sequence (amino acids −19 to1438 of SEQ ID NO:2; amino acids −19 to 2332 of SEQ ID NO:6; amino acids−19 to 740 of SEQ ID NO:8; amino acids −19 to 745 of SEQ ID NO:10; oramino acids −20 to 684 of SEQ ID NO:12). The Factor VIII (or Factor VIIIportion of a chimeric polypeptide) can be identical to a Factor VIIIamino acid sequence shown in Sequence Table 2 with a signal sequence(amino acids −19 to 1438 of SEQ ID NO:2; amino acids −19 to 2332 of SEQID NO:6; amino acids −19 to 740 of SEQ ID NO:8; amino acids −19 to 745of SEQ ID NO:10; or amino acids −20 to 684 of SEQ ID NO:12).

B. Factor IX Polypeptides

“Factor IX”, “FIX”, “protein having FIX activity”, “FIX protein”, or“FIX polypeptide” as used herein, means functional Factor IX polypeptidein its normal role in coagulation, unless otherwise specified. Thus, theterm Factor IX includes variant polypeptides that are functional and thepolynucleotides that encode such functional variant polypeptides. FactorIX polypeptides include the human, bovine, porcine, canine, feline, andmurine Factor IX polypeptides. The full length polypeptide andpolynucleotide sequences of Factor IX are known, as are many functionalvariants, e.g., fragments, mutants and modified versions. Factor IXpolypeptides include full-length Factor IX, full-length Factor IX minusMet at the N-terminus, full-length Factor IX minus the signal sequence,mature Factor IX (minus the signal sequence and propeptide), and matureFactor IX with an additional Met at the N-terminus. Factor IX can bemade by recombinant means (“recombinant Factor IX” or “rFIX”), i.e., itis not naturally occurring or derived from plasma.

great many functional Factor IX variants are known. Internationalpublication number WO 02/040544 A3, which is herein incorporated byreference in its entirety, discloses mutants that exhibit increasedresistance to inhibition by heparin at page 4, lines 9-30 and page 15,lines 6-31. International publication number WO 03/020764 A2, which isherein incorporated by reference in its entirety, discloses Factor IXmutants with reduced T cell immunogenicity in Tables 2 and 3 (on pages14-24), and at page 12, lines 1-27. International publication number WO2007/149406 A2, which is herein incorporated by reference in itsentirety, discloses functional mutant Factor IX molecules that exhibitincreased protein stability, increased in vivo and in vitro half-life,and increased resistance to proteases at page 4, line 1 to page 19, line11. WO 2007/149406 A2 also discloses chimeric and other variant FactorIX molecules at page 19, line 12 to page 20, line 9.

International publication number WO 08/118507 A2, which is hereinincorporated by reference in its entirety, discloses Factor IX mutantsthat exhibit increased clotting activity at page 5, line 14 to page 6,line 5. International publication number WO 09/051717 A2, which isherein incorporated by reference in its entirety, discloses Factor IXmutants having an increased number of N-linked and/or O-linkedglycosylation sites, which results in an increased half-life and/orrecovery at page 9, line 11 to page 20, line 2. Internationalpublication number WO 09/137254 A2, which is herein incorporated byreference in its entirety, also discloses Factor IX mutants withincreased numbers of glycosylation sites at page 2, paragraph [006] topage 5, paragraph [011] and page 16, paragraph [044] to page 24,paragraph [057]. International publication number WO 09/130198 A2, whichis herein incorporated by reference in its entirety, disclosesfunctional mutant Factor IX molecules that have an increased number ofglycosylation sites, which result in an increased half-life, at page 4,line 26 to page 12, line 6. International publication number WO09/140015 A2, which is herein incorporated by reference in its entirety,discloses functional Factor IX mutants that an increased number of Cysresidues, which can be used for polymer (e.g., PEG) conjugation, at page11, paragraph [0043] to page 13, paragraph [0053].

In addition, hundreds of non-functional mutations in Factor IX have beenidentified in hemophilia patients, many of which are disclosed in Table1, at pages 11-14 of International publication number WO 09/137254 A2,which is herein incorporated by reference in its entirety. Suchnon-functional mutations are not included in the invention, but provideadditional guidance for which mutations are more or less likely toresult in a functional Factor IX polypeptide.

The Factor IX (or Factor IX portion of a chimeric polypeptide) can be atleast 90% or at least 95% or 100% identical to a Factor IX amino acidsequence shown in Sequence Table 2 without a signal sequence andpropeptide sequence (amino acids 1 to 415 of SEQ ID NO:14), oralternatively, with a propeptide sequence, or with a propeptide andsignal sequence (full length Factor IX).

Factor IX coagulant activity is expressed as International Unit(s) (IU).One IU of Factor IX activity corresponds approximately to the quantityof Factor IX in one milliliter of normal human plasma. Several assaysare available for measuring Factor IX activity, including the one stageclotting assay (activated partial thromboplastin time; aPTT), thrombingeneration time (TGA) and rotational thromboelastometry (ROTEM®).

“Protein having FIX activity which is in its activated form,” or“activated FIX protein” means the activated form of a corresponding FIXprotein/polypeptide. The term “activated” in connection with anactivated FIX protein/polypeptide is used according to its commonmeaning. For example, in vivo. Factor IX is produced as a zymogen, aninactive precursor. It is processed to remove a signal peptide,glycosylated and then cleaved. e.g., by factor XIa or factor VIIa toproduce activated FIX (FIXa), a two-chain form where the two chains arelinked by a disulfide bridge. For example, activated FIX protein can beformed during the production and/or purification of a recombinant FIXprotein. In one example, in a pharmaceutical FIX polypeptidecompositions, the activated form of the FIX polypeptide can beconsidered an impurity.

C. Factor VIII and Factor IX Chimeric Polypeptides

“Chimeric polypeptide,” as used herein, means a polypeptide thatincludes within it at least two moieties (or portions thereof such assubsequences or peptides) from different sources. Chimeric polypeptidescan include two, three, four, five, six, seven, or more polypeptides orportions thereof from different sources, such as different genes,different cDNAs, or different animal or other species. Chimericpolypeptides can include one or more linkers joining the differentpolypeptides or portions thereof. Thus, the polypeptides or portionsthereof can be joined directly or they can be joined indirectly, vialinkers, or both, within a single chimeric polypeptide. Chimericpolypeptides can include additional peptides such as signal sequencesand sequences such as 6His and FLAG that aid in protein purification ordetection. In addition, chimeric polypeptides can have amino acid orpeptide additions to the N- and/or C-termini.

In certain aspects, a chimeric polypeptide is a long-acting clottingfactor. “Long-acting clotting factor” such as long-acting FVIII orlong-acting FIX is a Factor VIII or Factor IX having an increasedhalf-life (also referred to herein as t½, t½ beta, elimination half-lifeand HL) over a reference Factor VIII or a reference Factor IX,respectively. The increased half-life of a long-acting Factor VIII or along-acting Factor IX may be due to fusion to one or more non-FactorVIII or non-Factor IX polypeptides such as, e.g., Fc, XTEN, albumin, aPAS sequence, transferrin, CTP (28 amino acid C-terminal peptide (CTP)of hCG with its 4 O-glycans), polyethylene glycol (PEG), hydroxyethylstarch (HES), albumin binding polypeptide, albumin-binding smallmolecules, or two or more combinations thereof. The increased half-lifemay be due to one or more modification, such as, e.g., pegylation.Exemplary long-acting clotting factor polypeptides include. e.g.,chimeric Factor VIII polypeptides comprising Fc, chimeric Factor VIIIpolypeptides comprising XTEN, chimeric Factor VIII polypeptidescomprising albumin, chimeric Factor IX polypeptides comprising Fc,chimeric FIX polypeptide comprising XTEN, or chimeric Factor IXpolypeptide comprising albumin. Additional exemplary long-acting FactorVIII polypeptides include, e.g., pegylated Factor VIII or pegylatedFactor IX.

The “reference” polypeptide, in the case of a long-acting chimericFactor VIII polypeptide, is a polypeptide consisting essentially of theFactor VIII portion of the chimeric polypeptide, e.g., the same FactorVIII portion without the Fc portion, without the XTEN portion, orwithout the albumin portion. The “reference” polypeptide, in the case ofa long-acting chimeric Factor IX polypeptide, is a polypeptideconsisting essentially of the Factor IX portion of the chimericpolypeptide, e.g., the same Factor IX portion without the Fc portion,without the XTEN portion, or without the albumin portion. Likewise, thereference polypeptide in the case of a modified Factor VIII or Factor IXis the same Factor VIII or Factor IX without the modification,respectively. e.g., a Factor VIII without the pegylation or a Factor IXwithout the pegylation.

In some aspects, the chimeric polypeptide comprises a Factor VIIIportion and a non-Factor VIII portion. In some aspects, the chimericpolypeptide comprises a Factor IX portion and a non-Factor IX portion.Exemplary non-Factor VIII or non-Factor IX portions include, e.g., Fc,and albumin. Exemplary chimeric polypeptides include, e.g., chimericFactor VIII-Fc polypeptides, chimeric Factor IX-Fc polypeptides,chimeric Factor VIII-albumin polypeptides, and chimeric FactorIX-albumin polypeptides.

In some aspects, a chimeric polypeptide comprising a Factor VIII orFactor IX portion of a chimeric protein has an increased half-life(t1/2) over a polypeptide consisting of the same Factor VIII or FactorIX portion without the non Factor VIII or Factor IX portion. A chimericFactor VIII or Factor IX polypeptide with an increased t1/2 can bereferred to herein as a long-acting Factor VIII or Factor IX.Long-acting chimeric Factor VIII or Factor IX polypeptides include.e.g., Factor VIII or Factor IX fused to Fc (including. e.g., chimericFactor VIII or Factor IX polypeptides in the form of a hybrid such as aFVIIIFc monomer dimer hybrid; see e.g. FIGS. 1 and 2, and Table 2; andU.S. Pat. Nos. 7,404,956 and 7,348,004).

Exemplary chimeric Factor VIII polypeptides include, e.g., chimericFactor VIII-Fc polypeptides. Exemplary chimeric Factor VIII-Fcpolypeptides include. e.g., SEQ ID NOs:2, 6, 8, 10, and 12 (SequenceTable 2), with or without their signal sequences and the chimeric Fcpolypeptide of SEQ ID NO:4 (Sequence Table 2). The chimeric polypeptidecan comprise a sequence at least 90% or 95% identical to the Factor VIIIand Fc amino acid sequence shown in Sequence Table 2A(i) without asignal sequence (amino acids 1 to 1665 of SEQ ID NO:2) or at least 90%or 95% identical to the Factor VIII and Fc amino acid sequence shown inSequence Table 2A(i) with a signal sequence (amino acids −19 to 1665 ofSEQ ID NO:2). The chimeric polypeptide can comprise a sequence identicalto the Factor VIII and Fc amino acid sequence shown in Sequence Table2A(i) without a signal sequence (amino acids 1 to 1665 of SEQ ID NO:2)or identical to the Factor VIII and Fc amino acid sequence shown inSequence Table 2A(i) with a signal sequence (amino acids −19 to 1665 ofSEQ ID NO:2).

Exemplary chimeric Factor IX polypeptides are Factor IX-FcRn BP chimericpolypeptides, e.g., Factor IX-Fc chimeric polypeptides such as the FIXFcin SEQ ID NO:2 (Sequence Table 2), with or without its signal sequenceand propeptide. Other exemplary chimeric polypeptides include, but arenot limited to, Factor IX-XTEN chimeric polypeptides. Factor IX can befused to either N-terminus or C-terminus of XTEN. The chimericpolypeptide can comprise a sequence at least 90% or at least 95% or 100%identical to the Factor IX and FcRn BP, e.g., the Fc amino acid sequenceshown in Sequence Table 2A without a signal sequence and propeptidesequence (amino acids 1 to 642 of SEQ ID NO:14), or alternatively, witha propeptide sequence, or alternatively with a signal sequence and apropeptide sequence.

D. Factor VIII and Factor IX Hybrid Polypeptides

“Hybrid” polypeptides and proteins, as used herein, means a combinationof a chimeric polypeptide with a second polypeptide. The chimericpolypeptide and the second polypeptide in a hybrid can be associatedwith each other via non-covalent protein-protein interactions, such ascharge-charge or hydrophobic interactions. The chimeric polypeptide andthe second polypeptide in a hybrid can be associated with each other viacovalent bond(s) such as disulfide bonds. The chimeric peptide and thesecond peptide can be associated with each other via more than one typeof bond, such as non-covalent and disulfide bonds. Hybrids are describedin WO 2004/101740, WO2005/001025, U.S. Pat. No. 7,404,956, U.S. Pat. No.7,348,004, and WO 2006/074199, each of which is incorporated herein byreference in its entirety. The second polypeptide can be a second copyof the same chimeric polypeptide or it can be a non-identical chimericpolypeptide.

In some aspects, the second polypeptide is a polypeptide comprising anFc. In some aspects, the chimeric polypeptide is a chimeric FactorVIII-Fc polypeptide and the second polypeptide consists essentially ofFc, e.g, a rFVIIIFc recombinant fusion protein consisting of a singlemolecule of recombinant B-domain deleted human FVIII (BDD-rFVIII) fusedto the dimeric Fc domain of the human IgG1, with no intervening linkersequence. This hybrid polypeptide is referred to herein as FVIIIFcmonomeric Fe fusion protein, FVIIIFc monomer hybrid, monomeric FVIIIIFchybrid, and FVIIIFc monomer-dimer. In some aspects, the chimericpolypeptide is a Factor IX-FcRn BP, e.g., Factor IX-Fc chimericpolypeptide, and the second polypeptide consists essentially of Fc. See.e.g., Sequence Table 2 (SEQ ID NOs:14 and 4). See, e.g., U.S. Pat. No.7,404,956, which is incorporated herein by reference in its entirety.

The second polypeptide in a hybrid can comprise or consist essentiallyof a sequence at least 90% or at least 95%, or 100% identical to theamino acid sequence shown in Sequence Table 2 without a signal sequence(amino acids 1 to 227 of SEQ ID NO:4), or alternatively, at least 90%,or at least 95%, or 100% identical to the amino acid sequence shown inTable 2 with a signal sequence (amino acids −20 to 227 of SEQ ID NO:4).

Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention. All patents and publicationsreferred to herein are expressly incorporated by reference in theirentireties.

EXAMPLES Materials and Methods

Preparation of Test Strips (FVIII)

Disposable strips were the same type as currently used in Coag-Sense™PT/INR Monitoring System (CoaguSense, Inc, Fremont, Calif.) without theProthrombin Time reagents added to the strip. Strips were coated with1.25 μL of 80% of 0.1 mg/mL purified Factor IXa (obtained fromHaematologic Technologies, Essex Junction, Vt.) plus 20% phospholipidvesicles prepared as described below. Strips were air dried in a dry 37°C. incubator and individually sealed in plastic pouches containing adesiccant.

Preparation of Phospholipids

The strip for the Standard FMS Factor VII assay used an equal mix ofPhospholipid Blend 2 and Phospholipid Blend 8. Phospholipid Blend 2consisted of a phosphatidylcholine (PC) and phosphatidylserine (PS)mixture at a 70:30 molar ratio (mol-%). Phospholipid Blend 8 consistedof a PC. PS and phosphatidylglycerol (PG) mixture at a 80:10:10 molarratio. Thus, the optimized ratio of PL on the Standard FMS Factor VIIIassay strip (“standard strip”) was 75:20:5 (PC:PS:PG).

To prepare phospholipid blends, a total of 2.6 μmoles of phospholipidsdissolved in chloroform was dispensed into a glass tube, whereindividual phospholipids were mixed at the defined molar ratio(synthetic phospholipids can be obtained from Avanti Polar Lipids,Alabaster, Ala.). The phospholipid mixture was dried in a fume hoodunder a gentle stream of nitrogen or argon. When dry, phospholipidmixtures were dried in a speed-vac for an additional 1 hour to overnightunder high vacuum to remove any residual chloroform. 2.6 mL HepesBuffered Saline (10 mM HEPES pH 7.4 and 140 mM NaCl) at room temperaturewere added to the dried-down phospholipid mixture until all the driedlipid suspension was re-hydrated. The tube was incubated at 37° C. andvortexed intermittently. The result was a milky, uniform suspension.Small unilamellar vesicles were prepared by sonication for 7-10 minuteson ice with one-minute gap intervals between the shocks. The residuallarge vesicles were removed by filtering using 45-micron filters.

Test Procedure

The test strip containing the dried activator mixture (FIXa/PL mixture)was pre-warmed automatically after insertion into the measuring device.When the device was ready to receive a sample, patient plasma or wholeblood was recalcified with 0.3 volumes of 60 mM CaCl₂ and 12 μL of therecalcified sample were immediately added to the well of the pre-warmedtest strip. Clot formation was initiated as the blood/plasma dissolvedthe dried activator on the test strip. The device measured the time frominitiation to formation of a clot with defined characteristics. Thistime interval was referred to as clotting time (Ct). See, e.g., FIG. 3for an example of the application of the Standard FMS Factor VIII assay.

Further optimization of the FMS FVIII assay included the addition oftrace amounts of Factor VIII (approx. 1% of normal) to the driedactivator mixture, which resulted in a base clot time, rather than“timing out” in the absence any clot formation in severely hemophilicpatient samples. Further optimization of the FMS FVIII assays willinclude adding CaCl₂ on the dried test strip rather than off-striprecalcification of the sample.

Optimized FMS FVIII assay chemistry during the clot reaction can containthe following reactants (i) 9.2 μL patient blood; (ii) 2.8 μL buffer;(iii) 14 mM CaCl₂; (iv) 21 μM phospholipid mix (PC:PS:PG at 75:20:5);(v) 8.3 μg Factor IXa; and (vi) 24 pg Factor VIII.

FMS Factor IX Assay

The FMS Factor IX assay (see. e.g., FIG. 4 for an example of theapplication of the Standard FMS Factor IX assay) followed the sameprocedure as for Factor VIII, except that (i) the activator mixtureincluded on the test strip contains Factor XIa instead of Factor IXa(the exact amount of Factor XIa needed varied depending on the specificactivity of this reagent and is titrated for optimal amount); and, (ii)trace amounts of Factor IX could optionally be added to the strip toachieve a base clotting time to improve responsiveness to small amountsof Factor IX in the patient sample.

Example 1: Standard Factor Monitoring System (FMS) Assay

Both Standard FMS assays (Standard FMS Factor VIII assay and StandardFMS Factor IX assay) were initiated by the application of 12 μL ofrecalcified patient plasma directly to the test bed, a disposable teststrip containing activated coagulation factor-phospholipid complex. Onthis test bed, utilizing linear log-log curve fitting of concentrationversus clotting time, both Standard FMS assays performed well when anindividual Hemophilia A or Hemophilia B donor plasma was spiked witheither rFVIIIFc or rFIXFc, respectively (TABLE 1).

TABLE 1 Application of Standard FMS Factor VIII and Factor IX assays toHemophilia A and Hemophilia B Samples spiked with either rFVIIIFc orrFIXFc, respectively. Standard FMS Standard FMS Factor VIII Assay FactorIX Assay Assay Range (IU/dL) 0.8-100 0.2-100 Accuracy (% Spike Recovery)+/−10% +/−10.1 Precision (% CV) 3.8 1.9

Example 2: Sensitivity of the Standard FMS Assay to IndividualPhenotypic Variability

The disclosed “Standard FMS” assay system utilized undiluted patientplasma, thus, it was more analogous to an aPTT assay than to theone-stage factor assay. In the laboratory aPTT assay, one part undilutedpatient plasma is generally combined with equal parts of liquid aPTTreagent and of CaCl₂ solution. In contrast, in the one-stage assay, onepart diluted (1:5) patient plasma is generally combined with one partfactor deficient plasma, one part aPTT reagent, and one part CaCl₂.Because of this dilution of patient plasma with factor deficient plasma,the one stage factor assay largely masks inter-subject variability thatcan occur as a result of variable levels of non-target coagulationfactors (TABLE 2).

TABLE 2 Hemophilia Donor Phenotypic Variability VIII Deficient PlasmaSamples Sample PT (sec) APTT (sec) Fib. (mg/dL) % II % V % VII % VIII %IX % X % XI % XII HRF 11P2F8 11.1 104.8 293 85 86 80 <1 86 107 79 85 HRF11P3F8 11.2 >400 353 82 76 95 <1 98 95 82 87 GK 892-2086 11.6 103.0 24878 67 81 <1 76 93 80 126 HRF 10-389 11.2 117.2 276 78 94 72 <1 74 93 6280 HRF 10-1081 10.8 >400 334 89 116 80 <1 113 122 100 92 BD-001 11.070.2 293 In89 92 75 2.2 90 97 90 121 BD1-002 11.3 111.3 345 92 90 55 <1120 125 99 120 BD1-005 10.4 91.4 342 99 98 84 <1 128 132 115 105 BD-00X10.7 76.3 236 94 75 91 <1 102 101 83 85 IX Deficient Plasma SamplesSample PT (sec) APTT (sec) Fib. (mg/dL) % II % V % VII % VIII % IX % X %XI % XII HRF 11P1F9 12.0 105.6 383 85 111 57 91 2 114 94 110 HRF 09-86012.9 82.6 226 89 88 43 63 2 115 80 77 HRF 11-019 11.7 106.8 387 82 11160 101 2 114 105 113 GK 929-2074 11.8 113.1 357 85 113 58 94 2 117 97105 K 939-2054 12.1 97.8 257 89 96 53 72 2 130 79 89

Chromogenic based Factor VIII and Factor IX assays are also insensitiveto non-target coagulation factors because of the large sample dilutionsand physiologically irrelevant concentrations of added coagulationfactors and inhibitors.

To assess the sensitivity of the Standard FMS assay to individualphenotypic variability, four plasma samples collected from individualhemophilia A (HemA) donors after 5 day washout period (essentially 0%Factor VIII, confirmed by in-house assays) were spiked at 6 levels ofrFVIIIFc (100%, 50%, 25%, 12.5%, 6.3%, 3.1%) and the clotting time wasdetermined using the Standard FMS Factor VIII assay. The Standard FMSassay was applied using no equilibrating factors, and activation mixturecomprising FIXa and Phospholipid Blend 2 (FIG. 5A) or Phospholipid Blend8 (FIG. 5B). Although log-linear plots of rFVIIIFc concentration versusclot time for the 4 individuals displayed the same assay range andsensitivity, each of the individuals displayed a unique dose response toadded rFVIIIFc.

Example 3: Alternate FMS Assay

To eliminate the observed phenotypic variability, several modificationsto the Standard FMS methodology were investigated. Substitution of lesssensitive phospholipid blends was able to reduce phenotypic variability.Adding a variety of purified coagulation factors, e.g., Factors II, VII,VIII, IX, X, XI, XII, XII, fibrinogen, vWF and Tissue Factor, andinhibitors, e.g., CTI, aprotinin. ε-aminocaproic acid (EACA),D-Phenylalanyl-1-prolyl-1-arginine chloromethyl ketone-Factor VIIa(FPRCK-FVIIa), or anti-FVIII monoclonal antibodies, also was able toreduce phenotypic variability.

A variant of the Standard FMS was developed. This variant, referred toas the “Alternate FMS” assay throughout the instant disclosure wasessentially designed as a hybrid between the “Standard FMS” assay and aone stage coagulation factor assay. The Alternate FMS assay alsoutilized an activation mixture comprising activated coagulation factor(FIXa or FXIa) combined with a phospholipid vesicle preparation anddried on the solid substrate (e.g., a disposable test strip). In theplasma based Alternate FMS assay (see FIGS. 6A and 6B), one parthemophilia plasma spiked with either rFVIIIFc or rFIXFc was mixed withthree parts of a substrate plasma that had been depleted of the assaytarget factor. In this manner, the variability of non-target plasmacomponents was normalized by addition of the substrate plasma.

This combination of hemophilia plasma and substrate plasma was done inan all-liquid system resulting in a four-fold dilution of the hemophiliatest plasma, thus increasing the lower level of detection.

TABLE 3 Range, average precision and accuracy data for Alternate FMSFactor VIII and Factor IX Assays Alternate FMS Alternate FMS Factor VIIIAssay Factor IX Assay Range (IU/dL) 1.5-100 0.4-100 Average Precision (%CV) 1.8% 1.0% Accuracy (% Spike Recovery) +/−10.2 +/−11.3

It is anticipated that preparing a dry substrate plasma format willsignificantly improve the lower level of detection, since the targetanalyte will be four-fold higher than in the current format.

Example 4: Evaluation of Inter-Subject Variability Using the AlternateFMS Assay

To evaluate the ability of the Alternate FMS assay format to decreaseinter-subject variability, 14 hemophilia A and 9 hemophilia B plasmasamples were procured to conduct spike recovery studies. Plasma sampleswere obtained from 4 different vendors, collected by 3 differentmethodologies on 3 different anticoagulants. Plasma samples includedimmunodepleted as well as congenital hemophilia plasma. Plasma sampleswere also subjected to different storage conditions as well as freezethaw cycles. The effect of a single plasma freeze thaw cycle onAlternate FMS assay performance is shown in FIGS. 7A (Alternate FMSFactor IX assay) and 7B (Alternate FMS Factor VIII assay).

In each case, samples contained 12 μL of re-calcified plasma mixed 1:3with substrate plasma (Factor IX deficient plasma supplemented withdefined levels of rFIXFc in the Alternate FMS Factor IX assay; andFactor VIII deficient plasma supplemented with defined levels ofrFVIIIFc in the Alternate FMS Factor VIII assay). The results of thespike recovery studies are summarized in FIGS. 8A and 8B.

The samples presented in FIG. 8A were citrated plasma samples collectedfrom 14 hemophilia A donors (assumed <1% Factor VIII). Plasma sampleswere collected at 3 sites, using 3 different methodologies and spikedwith varying levels of rFVIIIFc. Each of these samples was also assayedon a laboratory reference system (MLA-1000 Coagulation Analyzer)utilizing the manufacturer's suggested reagents and calibration standardplasma traceable to the appropriate WHO standard. For hemophilia Aplasmas spiked with rFVIIIFc, the theoretical values (assuming FactorVIII plasma levels supplied by vendors) were not in good agreement withvalues determined on the MLA. This could be related to sample qualityissues, errors in the MLA assay, or errors in stock Factor VIIIconcentrations. For this reason, spike recovery assays for Alternate FMSFactor VIII assay in FIG. 8A were plotted as MLA value vs. Alternate FMSFactor VIII assay value (FIG. 8A). The dashed lines on the graphrepresented +/−20% of the MLA determined Factor VIII level. The averageCV for all the Alternate FMS Factor VIII assays performed on the 14hemophilia A donors was 3.1.

The samples presented in FIG. 8B were citrated plasma samples collectedfrom 9 hemophilia B donors (8 donors were <1% Factor IX; 1 donor was˜34% Factor IX). Plasma samples were collected at 3 sites, using 3different methodologies and spiked with varying levels of rFIXFc. Eachof these samples was also assayed on a laboratory reference system(MLA-1000 Coagulation Analyzer) utilizing the manufacturer's suggestedreagents and calibration standard plasma traceable to the appropriateWHO standard. Factor IX values and MLA values were in good agreement.Assuming the MLA value was the “true value.” a plot was constructed ofMLA factor concentration versus Alternate FMS Factor IX assayconcentrations (FIG. 83). The dashed lines on the graph represented+/−20% of the MI. A determined Factor IX level. The average CV for the64 determinations on 8 different instruments was 3.1% (range 0-10.2%).There was no apparent bias over the range of Factor IX concentrations(1.5-12.5 IU/dL) tested.

Assuming that the concentration determined by the MLA assay was the truevalue, then 61% of the Alternate FMS Factor VIII determinations and 75%of the Alternate FMS Factor IX determinations were within a +/−20%accuracy range. The average spike recovery for the Alternate FMS FactorVIII assay was +/−21% (Range 0.8-51%) and for the Alternate FMS FactorIX assay it was +/−23% (Range 0.3-98%). Assay performance was remarkableconsidering the non-ideal nature of the frozen plasma samples,uncertainties surrounding stock concentrations of rFVIII and rFIXproducts, not yet optimized test bed parameters, manual solid substrateproduction, and non-standardized “off the shelf” critical raw materials.

Example 5: Adaptation of Alternate FMS Assay to Whole Blood Samples

The feasibility of adapting these Alternate FMS assays to whole bloodwas examined in normal donors and in hemophilia A (n=4) (results shownin Example 5.1) and hemophilia B (n=1) subjects (results shown inExample 5.2).

The experimental protocol was similar for each round of testing usingAlternate FMS Factor VIII and Factor IX assays. In both cases, subjectswere asked to suspend factor replacement therapy for a minimum of 4 daysprior to the test date. Citrated venous whole blood and fingerstickwhole blood samples were collected prior to patient self-administrationof their individual routine replacement therapy. A second set ofcitrated venous whole blood and fingerstick whole blood samples werecollected and tested 20-40 minutes post infusion. Fingerstick samples,pre and post infusion were applied directly to the FMS test bed (solidsubstrate containing activation mixture). Aliquots of pre-infusioncitrated venous blood samples were spiked with varying levels of theappropriate drug product (either rFVIIIFc or rFIXFc) and then assayedusing the Alternate FMS assay. Samples were also tested on the MLAsystem.

Example 5.1: Adaptation of Alternate FMS Factor VIII Assay to WholeBlood Samples

The Alternate FMS Factor VIII assay was applied to six hemophilia Asubjects. The results of the last 2 test events are summarized in FIGS.9-14 and TABLES 4 and 5. A direct comparison of the data in FIGS. 9-12and FIG. 14 test data cannot be made owing to reagent lot changes. Themajor goal of the testing was to determine the feasibility of theAlternate FMS assay to mute the inter-subject variability inherent inthe Standard FMS assay in whole blood samples from hemophilia A donorsas it did in frozen plasma samples.

FIGS. 9A and 9B summarize an experiment in which citrated venous andfingerstick whole blood samples were collected from 2 hemophilia Asubjects 5 days pre and post infusion treatment with factor replacementtherapy. Samples were spiked with increasing concentrations (0 IU/dl to200 IU/dL) of rFVIIIFc and tested utilizing the Standard FMS standardand Alternate FMS assays. Assuming both subjects' baseline FVIII valueswere 0 IU/dL, then the 2 hemophilia A subjects displayed disparate doseresponse in the Standard FMS assay. This was likely due to inter-subjectvariability in coagulation factors other than FVIII or to cellularcomponents. When these same samples were run using the Alternate FMSassay, both subjects displayed similar concentration dependent clottimes over the range of 1.5-200 IU/dL rFVIIIFc.

Each of the venous whole blood samples from the spike experimentsdescribed above was centrifuged to prepare plasma samples. The frozenplasma retentions were subsequently assayed on the MLA system (FIGS.10A, 10B and 10C), using the Standard FMS Factor VIII assay (FIGS. 11Aand 11B), and using the Alternate FMS Factor VIII assay (FIGS. 12A and12B) to ascertain the relationship between FMS whole blood clot timesversus plasma clot times. The trends in dose response for plasma samplesmirrored those of venous whole blood samples (see FIG. 13) indicating acorrelation that can be exploited for calibration purposes.

Example 5.2: Application of the Alternate FMS Factor VIII Assay toCitrated and Non-Citrated Whole Blood Samples

Since the ultimate goal for the FMS assays was to use fingerstick wholeblood that was not citrate anticoagulated, a comparison between citratedvenous blood, citrated plasma and non-citrated fingerstick blood wasperformed in parallel to the previously described experiment. Theresults of this comparison are summarized in TABLE 4. Fingerstick wholeblood samples displayed concentration-dependent clotting analogous tovenous blood and plasma samples in both the Standard and the AlternateFMS methods.

TABLE 4 FMS FVIII Assays - Plasma/Venous/Fingerstick Blood SamplingCorrelation Standard FMS Alternate FMS Factor VIII Assay Factor VIIIAssay DONOR ID 003 005 003 005 Pre-Dose (<1% FVIII) Clot Time (seconds)Venous >300 >300 116.5 112.6 Fingerstick >300 >300 ND NDPlasma >300 >300 102.6 104.9 Venous + 100% rFVIII-Fc 61.5 55.2 77.8 76.3Spike Plasma + 100% rFVIII-Fc 45.8 46.5 64.4 64 Spike Post-Dose (assume100% FVIII) Venous 65 56.1 89.1 83.6 Fingerstick 54.7 48.6 71.1 NDPlasma 52.9 47 72.2 70.4 ND = Not determined

Two additional Hemophilia A donors were tested using essentially thesame protocol with the following changes: (i) testing focused mainly onthe Alternate FMS assay, (ii) new lots of strips and raw materials,including substrate plasma, were used in this testing, and (iii) amodified fingerstick protocol using a high flow pediatric lancet wasemployed. Also, plasma samples separated from the citrated venous bloodwere assayed prior to freezing and retesting. Samples were spiked withincreasing levels (0.8-200 IU/dL) rFVIIIFc prior to assay.

The results of this second round of testing are displayed in FIGS. 14Aand 14B, and TABLE 5. General assay performance was improved in thesetests compared with the results from the previous round of testing shownin TABLE 4.

TABLE 5 Alternate FMS Factor VIII Assay - Plasma/Venous/FingerstickBlood Sampling Correlation Alternate Method DONOR ID BD1-002 BD1-004Pre-Dose Samples CLOT TIME (seconds) Venous Blood >300 >300 FingerstickBlood >300 >300 Plasma >300 >300 Venous + 100% rFVII-Fc Spike 56.7 57.4Plasma + 100% rFVIII-Fc Spike 60.0 57.9 Post Dose Samples Venous Blood54.9 59.9 Fingerstick Blood Drop 1 53.6 55.1 Drop 2 57.7 61.7 Plasma,Neat 58.5 ND

Results indicated that, in the current format, the useful range for theAlternate FMS Factor VIII assay in venous blood is 1.5 IU/dL-200 IU/dL.The average CV for all of these determinations (n=17) was 2.1% (range0.3-4.8) with no trend in CV with level of FVIII. As it was designed todo, the Alternate FMS Factor VIII assay displayed minimized intersubjectvariability thought to result from natural variations in non-Factor VIIIeffectors.

It is anticipated that optimization of instrument parameters to improveclot detection at the lower (<1%) range will expand the useful range ofthe assay to <0.5 IU/dL. Limited data on fingerstick whole bloodindicated a good correlation to citrated whole blood and plasma.Experiments that follow temporal FMS fingerstick assays on post factorreplacement therapy will be used to explore useful range for thisformat.

Example 5.3: Adaptation of Alternate FMS Factor IX Assay to Whole BloodSamples

The utility of the Alternate FMS Factor IX assay in whole blood sampleswas demonstrated by performing experiments analogous to those describedfor Factor VIII on a Hem B subject. The Hem B subject suspended FactorIX replacement therapy for 4 days prior to the test date. Citratedvenous whole blood, citrated plasma, and fingerstick whole blood sampleswere collected pre- and post-self administration of the subject'sroutine Factor IX replacement therapy.

Aliquots of pre-infusion venous blood were spiked with increasing levelsof rFIXFc (0-200 IU/dL) for use in constructing dose-response curvesusing the Alternate FMS Factor IX assay. Plasma samples separated fromthese samples were assayed fresh using the Alternate FMS IX assay.Frozen retentions were subsequently assayed using the Alternate FMSFactor IX assay and the MLA reference system. The results from theseexperiments are summarized in FIGS. 15A and 15B, and TABLE 6. Theaverage CV for the 44 determinations performed on 8 random instrumentswas 1.7%. The range of CV values (0-5.8%) did not appear correlated toFactor IX levels.

The potential range in whole blood as demonstrated by the venous blooddose-response curves was 0.4 IU/dL-100 IU/dL with an average CV of 1.8%.Again, CVs were not significantly different over the entire range of theassay.

TABLE 6 Alternate FMS Factor IX Assay - Plasma/Venous/Fingerstick BloodSampling Correlation Alternate Method Factor IX Assay DONOR ID BD2-002Pre-Dose Samples Clot Time (seconds) Venous Blood 90.4 Fingerstick Blood103.5 Plasma 107.6 Venous + 100% rFVIII-Fc Spike 52.2 Plasma + 100%rFVIII-Fc Spike 53.8 Post Dose Samples Venous Blood 54.1 FingerstickBlood 62.8 Plasma, Neat 63.4

Example 6: Evaluation of Instrument-to-Instrument Variability

This experiment used 8 instruments in random order for 64 determinationsusing the Hem A samples used in Example 4. Instrument to instrumentvariability was evaluated by assaying a single Factor VIII deficientplasma sample spiked to 100% and 3% rFVIIIFc in duplicate on 16 researchinstruments (FIG. 16). The observed CV values for the 100% spike (1.7%)and the observed CV value for the 3% spike (3.8%) were exceptional inlight of the fact that no tuning was done on those instruments and theresults included inter-instrument results.

Example 7: Factor Monitoring Vs. PK Determination Vs. Global HemostasisTest

Accurate clotting factor level determination in patients is a technicalchallenge. Several approaches can be use, e.g., factor monitoring,pharmacokinetics (PK) determination, or using a global hemostasis test.

Factor monitoring can be accomplished by routine measurement of FactorVIII or Factor IX levels by finger stick at treatment center and/or atthe patient's home. This approach has some advantages, e.g., it can beused for determining “traditional” PK (recovery, clearance, terminal t½,etc.), it allows long-term use by the patient or caregiver to evaluatecoverage at any given time, patients and health care providers arefamiliar with the concept of using factor levels for dosing (or theconcept is easy to adopt), and it can be used for any current FactorVIII or Factor IX products anywhere (“lab access” in developingcountries, diagnostic tool). The main drawback of this approach is thatit requires high accuracy and precision, which makes it the mosttechnically challenging approach. This is due, among other factor, tohigh inter-patient variability of coagulation time at equivalent factorlevels, which needs to be “equilibrated” or alternatively can requireone-time patient-specific calibration (e.g., single lab measurementduring training visit).

If using PK determination, the readout is clot time only; thus, it doesnot provide actual clotting factor levels. The main advantages of thisapproach are that it requires precision only, since accuracy for factorlevels is not needed, and no patient-specific calibration needed. Ingeneral, home-use with 5-8 measurements is likely to provide moreaccurate PK than 1-2 laboratory tests. This approach is also lesstechnically challenging than factor monitoring because inter-patientvariability in coagulation is not relevant for calculation of terminalt½, high precision and linearity of dose response have been shown forplasma, and proof of Concept for device chemistry has been achieved. Themain drawbacks of this approach are (i) lack of transparency for dosedetermination based on clot time, and (ii) long-term use by subjectrequires adoption of “global hemostasis” concepts, e.g., “Minimal clottime needed for individual hemostasis” (however, these concepts can beintuitive: “If your blood does not clot in 90 sec. you need morefactor”).

Determining Factor Level from Clot Time (Ct)

FIG. 17 exemplifies the use of clotting time measurements from FMSassays to determine factor levels. FMS assay results showed a linearrelationship between Ct and factor levels, which can be representedaccording to Equation 1:Ct=A×Ln(% Factor)+B  [Equation 1]where the slope A was similar for all patients, at the offset B was dueto patient-specific global coagulation differences.

It is possible to optimize the chemistry of the FMS assay (e.g., teststrip design) to eliminate B, so there is no patient-specific offset.This approach can be used to design “ready to use” factor monitoringdevices that do not require patient-specific calibration. Alternatively,the factor monitoring device can be customized for each patient bycalculating B for each patient and configuring the monitoring deviceaccordingly.

Determining Dosing Regimen Based on Clot Time (Ct)

FIG. 18 exemplifies how dosage regimens can be determined based onclotting time (Ct) determined using an FMS assay and data frompopulation modeling, e.g., based on the A-LONG rFVIIIFc clinical trial(ClinicalTrials.gov Identifier NCT01181128) or the B-LONG rFIXFcclinical trial (ClinicalTrials.gov Identifier NCT01027364).

Half-life (HL) in terminal phase of PK can be determined according tothe following set of equations:Ct₁ =A×Ln(% F ₁)+B  [from Equation 1]Ct₂ =A×Ln(% F ₂)+B  [from Equation 1]HL=−0.693×(T ₂ −T ₁)/Ln(% F ₁/% F ₂)  [Equation 3]hence:HL=−0.693×(T ₂ −T ₁)×A/(Ct₁−Ct₂)  [Equation 4]where A is the slope, a device-specific parameter which would be thesame for all patients. Note that the “offset” (B) becomes irrelevant,i.e., inter-patient differences in global coagulation do not affectterminal half-life.

Population modeling based on clinical trials can be used to calculateproduct-specific in vivo recovery and α-phase half-life (distributionphase half-life), which in turn can be used to calculate “time totrough.” FMS assay derived patient-specific half-life values and “timeto trough” values can in turn be used to calculate patient-specific doseand dose interval.

Global Hemostasis Test Based on FMS Assay

The FMS assays described above have been used to develop a globalhemostasis test. The FMS assays disclosed herein measure an individual'soverall clotting potential at any given level of coagulation factor.Proof of concept, sensitivity and range of the FMS assays have beenestablished as shown in the examples above. Furthermore, no externaldilution is performed as there is no need to equilibratepatient-specific differences.

As shown above in Equation 3, an increase in Ct as clotting factor iscleared over time correlates with terminal half-life (HL). Individualclot time at trough (Ct_(trough)) is a critical parameter to establishin each patient. With known patient-specific HL and Ct_(trough), the“time to trough” (T) after any measured Ct is then predictable and canbe calculated according to the following formula:T=−1.44×HL/(A×(Ct_(measured)−Ct_(trough))  [Equation 5]The time to trough (T) would correspond to the time to the next dose.

CONCLUSIONS

If the primary purpose of the application of the FMS assay is to definethe initial dosing regimen, a precise readout of clot time issufficient. There is no need for accuracy or patient-specificcalibration for terminal t½. To determine other PK parameters (e.g.,recovery and clearance) in individual patients, accurate factor levelsare required, however.

“Minimal clot time needed for hemostasis” can be an important biomarkerfor the individual patient. Once the FMS assay has been applied tomeasure (i) individual Ct at trough and (ii) change in Ct over time(i.e., individual terminal PK), estimating the “time to trough” (time tonext dose) based on a single Ct measurement is expected to be veryaccurate.

It is to be appreciated that the Detailed Description section, and notthe Summary and Abstract sections, is intended to be used to interpretthe claims. The Summary and Abstract sections can set forth one or morebut not all exemplary aspects of the present invention as contemplatedby the inventor(s), and thus, are not intended to limit the presentinvention and the appended claims in any way.

The present invention has been described above with the aid offunctional building blocks illustrating the implementation of specifiedfunctions and relationships thereof. The boundaries of these functionalbuilding blocks have been arbitrarily defined herein for the convenienceof the description. Alternate boundaries can be defined so long as thespecified functions and relationships thereof are appropriatelyperformed.

The foregoing description of the specific aspects will so fully revealthe general nature of the invention that others can, by applyingknowledge within the skill of the art, readily modify and/or adapt forvarious applications such specific aspects, without undueexperimentation, without departing from the general concept of thepresent invention. Therefore, such adaptations and modifications areintended to be within the meaning and range of equivalents of thedisclosed aspects, based on the teaching and guidance presented herein.It is to be understood that the phraseology or terminology herein is forthe purpose of description and not of limitation, such that theterminology or phraseology of the present specification is to beinterpreted by the skilled artisan in light of the teachings andguidance.

The breadth and scope of the present invention should not be limited byany of the above-described exemplary aspects, but should be defined onlyin accordance with the following claims and their equivalents.

SEQUENCE TABLE 1 Polynucleotide Sequences: FIX-FcA. B-Domain Deleted FVIIIFcA(i). B-Domain Deleted FVIIIFc Chain DNA Sequence (FVIII signal peptide underlined, Fcregion in bold) (SEQ ID NO: 1, which encodes SEQ ID NO: 2)

ggtgcagtggaactgtcatgggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacacctcagtcgtgtacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatccaggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtatcctactggaaagcttctgagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcctcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagtcaatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattggaatgggcaccactcctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaaacactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcttatgtcaaagtagacagctgtccagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttgatgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcggattggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggggaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaagatgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatggagagagatctagcttcaggactcattggccctctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaagaggaatgtcatcctgttttctgtatttgatgagaaccgaagctggtacctcacagagaatatacaacgctttctccccaatccagctggagtgcagcttgaggatccagagttccaagcctccaacatcatgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggtacattctaagcattggagcacagactgacttcctttctgtcttcttctctggatataccttcaaacacaaaatggtctatgaagacacactcaccctattcccattctcaggagaaactgtcttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgaccgccttactgaaggtttctagttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatacttgctgagtaaaaacaatgccattgaaccaagaagcttctctcaaaacccaccagtcttgaaacgccatcaacgggaaataactcgtactactcttcagtcagatcaagaggaaattgactatgatgataccatatcagttgaaatgaagaaggaagattttgacatttatgatgaggatgaaaatcagagcccccgcagctttcaaaagaaaacacgacactattttattgctgcagtggagaggctctgggattatgggatgagtagctccccacatgttctaagaaacagggctcagagtggcagtgtccctcagttcaagaaagttgttttccaggaatttactgatggctcctttactcagcccttataccgtggagaactaaatgaacatttgggactcctggggccatatataagagcagaagttgaagataatatcatggtaactttcagaaatcaggcctctcgtccctattccttctattctagccttatttcttatgaggaagatcagaggcaaggagcagaacctagaaaaaactttgtcaagcctaatgaaaccaaaacttacttttggaaagtgcaacatcatatggcacccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctggaaaaagatgtgcactcaggcctgattggaccccttctggtctgccacactaacacactgaaccctgctcatgggagacaagtgacagtacaggaatttgctctgtttttcaccatctttgatgagaccaaaagctggtacttcactgaaaatatggaaagaaactgcagggctccctgcaatatccagatggaagatcccacttttaaagagaattatcgcttccatgcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatcaaaggattcgatggtatctgctcagcatgggcagcaatgaaaacatccattctattcatttcagtggacatgtgttcactgtacgaaaaaaagaggagtataaaatggcactgtacaatctctatccaggtgtttttgagacagtggaaatgttaccatccaaagctggaatttggcgggtggaatgccttattggcgagcatctacatgctgggatgagcacactttttctggtgtacagcaataagtgtcagactcccctgggaatggcttctggacacattagagattttcagattacagcttcaggacaatatggacagtgggccccaaagctggccagacttcattattccggatcaatcaatgcctggagcaccaaggagcccttttettggatcaaggtggatctgttggcaccaatgattattcacggcatcaagacccagggtgcccgtcagaagttctccagcctctacatctctcagtttatcatcatgtatagtcttgatgggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttctttggcaatgtggattcatctgggataaaacacaatatttttaaccctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgcagcactcttcgcatggagttgatgggctgtgatttaaatagttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattactgcttcatcctactttaccaatatgtttgccacctggtctccttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggtgaataatccaaaagagtggctgcaagtggacttccagaagacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttaccagcatgtatgtgaaggagttcctcatctccagcagtcaagatggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcagggaaatcaagactccttcacacctgtggtgaactctctagacccaccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccagattgccctgaggatggaggttctgggctgcgaggcacaggacctctacgacaaaactcacacatgcccaccgtgcccagctccagaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaA(ii). Fc DNA sequence (mouse Igκsignal peptide underlined) (SEQ ID NO: 3, which encodes SEQ ID NO: 4)

ccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa B. Full Length FVIIIFcB(i). Full Length FVIIIFc DNA Sequence (FVIII signal peptide underlined, Fc region in bold)(SEQ ID NO: 5, which encodes SEQ ID NO: 6)

ggtgcagtggaactgtcatgggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacacctcagtcgtgtacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatccaggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtatcctactggaaagcttctgagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcctcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagtcaatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattgaaatgggcaccactcctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaaacactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcttatgtcaaagtagacagctgtccagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttgatgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcggattggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggggaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaagatgggccaactaaatcagatcctcgg

gatgggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttctttggcaatgtggattcatctgggataaaacacaatatttttaaccctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgcagcactcttcgcatggagttgatgggctgtgatttaaatagttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattactgcttcatcctactttaccaatatgtttgccacctggtctccttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggtgaataatccaaaagagtggctgcaagtggacttccagaagacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttaccagcatgtatgtgaaggagttcctcatctccagcagtcaagatggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcagggaaatcaagactccttcacacctgtggtgaactctctagacccaccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccagattgccctgaggatggaggttctgggctgcgaggcacaggacctctacgacaaaactcacacatgcccaccgtgcccagctccagaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaC(i). Heavy Chain (HC)-Fc DNA sequence (no linker between HC and Fe) (signal peptideunderlined, Fc region in bold) (SEQ ID NO: 7, which encodes SEQ ID NO: 8)

ggtgcagtggaactgtcatgggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacacctcagtcgtgtacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatccaggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtatcctactggaaagcttctgagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcctcattggagccctactagtatgtagagagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagtcaatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattggaatgggcaccactcctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaaacactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagcttatgtcaaagtagacagctgtccagaggaaccccaactacgaatgaaaaataatgaagaagcggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttgatgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccctcagcggattggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggggaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaagatgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatggagagagatctagcttcaggactcattggccctctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaagaggaatgtcatcctgttttctgtatttgatgagaaccgaaactggtacctcacagagaatatacaacgctttctccccaatccagctggagtgcagcttgaggatccagagttccaagcctccaacatcatgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggtacattctaagcattggagcacagactgacttcctttctgtcttcttctctggatataccttcaaacacaaaatggtctatgaagacacactcaccctattcccattctcaggagaaactgtcttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgaccgccttactgaaggtttctagttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatacttgctgagtaaaaacaatgccattgaaccaagagacaaaactcacacatgcccaccgtgcccagctccagaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctcattattcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaC(ii). Heavy Chain (HC)-Fc DNA sequence (5 amino acid linker between HC and Fc) (signalpeptide underlined, Fc region in bold, 5 amino acid linker is double-underlined) (SEQ ID NO: 9,which encodes SEQ ID NO: 10)

ggtgcagtggaactgtcatgggactatatgcaaagtgatctcggtgagctgcctgtggacgcaagatttcctcctagagtgccaaaatcttttccattcaacacctcagtcgtgtacaaaaagactctgtttgtagaattcacggatcaccttttcaacatcgctaagccaaggccaccctggatgggtctgctaggtcctaccatccaggctgaggtttatgatacagtggtcattacacttaagaacatggcttcccatcctgtcagtcttcatgctgttggtgtatcctactggaaagcttctgagggagctgaatatgatgatcagaccagtcaaagggagaaagaagatgataaagtcttccctggtggaagccatacatatgtctggcaggtcctgaaagagaatggtccaatggcctctgacccactgtgccttacctactcatatctttctcatgtggacctggtaaaagacttgaattcaggcctcattggagccctactagtatgtagagaagggagtctggccaaggaaaagacacagaccttgcacaaatttatactactttttgctgtatttgatgaagggaaaagttggcactcagaaacaaagaactccttgatgcaggatagggatgctgcatctgctcgggcctggcctaaaatgcacacagtcaatggttatgtaaacaggtctctgccaggtctgattggatgccacaggaaatcagtctattggcatgtgattggaatgggcaccactcctgaagtgcactcaatattcctcgaaggtcacacatttcttgtgaggaaccatcgccaggcgtccttggaaatctcgccaataactttccttactgctcaaacactcttgatggaccttggacagtttctactgttttgtcatatctcttcccaccaacatgatggcatggaagattatgtcaaagtagacagctgtccagaggaaccccaactacgaatgaaaaataatgaagaagaggaagactatgatgatgatcttactgattctgaaatggatgtggtcaggtttgatgatgacaactctccttcctttatccaaattcgctcagttgccaagaagcatcctaaaacttgggtacattacattgctgctgaagaggaggactgggactatgctcccttagtcctcgcccccgatgacagaagttataaaagtcaatatttgaacaatggccatcagaggattggtaggaagtacaaaaaagtccgatttatggcatacacagatgaaacctttaagactcgtgaagctattcagcatgaatcaggaatcttgggacctttactttatggggaagttggagacacactgttgattatatttaagaatcaagcaagcagaccatataacatctaccctcacggaatcactgatgtccgtcctttgtattcaaggagattaccaaaaggtgtaaaacatttgaaggattttccaattctgccaggagaaatattcaaatataaatggacagtgactgtagaagatgggccaactaaatcagatcctcggtgcctgacccgctattactctagtttcgttaatatggagagagatctagcttcaggactcattggccctctcctcatctgctacaaagaatctgtagatcaaagaggaaaccagataatgtcagacaagaggaatgtcatcctgttttctgtatttgatgagaaccgaagctggtacctcacagagaatatacaacgctttctccccaatccagctggagtgcagcttgaggatccagagttccaagcctccaacatcatgcacagcatcaatggctatgtttttgatagtttgcagttgtcagtttgtttgcatgaggtggcatactggtacattctaagcattggagcacagactgacttcctttctgtattattctctggatataccttcaaacacaaaatggtctatgaagacacactcaccctattcccattctcaggagaaactgtcttcatgtcgatggaaaacccaggtctatggattctggggtgccacaactcagactttcggaacagaggcatgaccgccttactgaaggtttctagttgtgacaagaacactggtgattattacgaggacagttatgaagatatttcagcatacttgctgagtaaaaacaat

ggaccgtcagtattcctcttccacccaaaacccaaggacaccatcatgatctccaggaccactgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtatccaacaaagccatcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgaccccatccagggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctaccgtgttggactccgacggctcattattcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtattctcatgctccgtgatacatgaggctctgcacaaccactacacgcagaagagcctctccctgtatccgggtaaa

gaaaccaaaacttacttttggaaagtgcaacatcatatggcacccactaaagatgagtttgactgcaaagcctgggcttatttctctgatgttgacctggaaaaagatgtgcactcaggcctgattggaccccttctggtctgccacactaacacactgaaccctgctcatgggagacaagtgacagtacaggaatttgctctgtttttcaccatctttgatgagaccaaaagctggtacttcactgaaaatatggaaagaaactgcagggctccctgcaatatccagatggaagatcccacttttaaagagaattatcgcttccatgcaatcaatggctacataatggatacactacctggcttagtaatggctcaggatcaaaggattcgatggtatctgctcagcatgggcagcaatgaaaacatccattctattcatttcagtggacatgtgttcactgtacgaaaaaaagaggagtataaaatggcactgtacaatctctatccaggtgtttttgagacagtggaaatgttaccatccaaagctggaatttggcgggtggaatgccttattggcgagcatctacatgctgggatgagcacactttttctggtgtacagcaataagtgtcagactcccctgggaatggcttctggacacattagagattttcagattacagcttcaggacaatatggacagtgggccccaaagctggccagacttcattattccggatcaatcaatgcctggagcaccaaggagcccttttcttggatcaaggtggatctgttggcaccaatgattattcacggcatcaagacccagggtgcccgtcagaagttctccagcctctacatctctcagtttatcatcatgtatagtcttgatgggaagaagtggcagacttatcgaggaaattccactggaaccttaatggtcttctttggcaatgtggattcatctgggataaaacacaatatttttaaccctccaattattgctcgatacatccgtttgcacccaactcattatagcattcgcagcactcttcgcatggagttgatgggctgtgatttaaatagttgcagcatgccattgggaatggagagtaaagcaatatcagatgcacagattactgcttcatcctactttaccaatatgtttgccacctggtctccttcaaaagctcgacttcacctccaagggaggagtaatgcctggagacctcaggtgaataatccaaaagagtggctgcaagtggacttccagaagacaatgaaagtcacaggagtaactactcagggagtaaaatctctgcttaccagcatgtatgtgaaggagttcctcatctccagcagtcaagatggccatcagtggactctcttttttcagaatggcaaagtaaaggtttttcagggaaatcaagactccttcacacctgtggtgaactctctagacccaccgttactgactcgctaccttcgaattcacccccagagttgggtgcaccagattgccctgaggatggaggttctgggctgcgaggcacaggacctctacgacaaaactcacacatgcccaccgtgcccagctccagaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaaD. FIX-Fc Chain DNA Sequence (SEQ ID NO: 13, which encodes SEQ ID NO: 14)pSYN-FIX-030 Nucleotide sequence (nt 1 to 7583):FIX exon 1 (signal peptide, 1st amino acid propeptide): nt 690-777FIX mini intron: nt 778-1076 FIX propeptide sequence: nt 1077-1126Mature FIX sequence: nt 1127-2371 Fc: nt 2372-3052gcgcgcgttgacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcttactggcttatcgaaattaatacgactcactatagggagacccaagcttcgcgacgtacggccgccaccatgcagcgcgtgaacatgatcatggcagaatcaccaggcctcatcaccatctgccttttaggatatctactcagtgctgaatgtacaggtttgtttccttttttaaaatacattgagtatgcttgccttttagatatagaaatatctgatgctgtcttcttcactaaattttgattacatgatttgacagcaatattgaagagtctaacagccagcacgcaggttggtaagtactgtgggaacatcacagattttggctccatgccctaaagagaaattggctttcagattatttggattaaaaacaaagactttcttaagagatgtaaaattttcatgatgttttcttttttgctaaaactaaagaattattcttttacatttcagtttttcttgatcatgaaaacgccaacaaaattctgaatcggccaaagaggtataattcaggtaaattggaagagtttgttcaagggaatctagagagagaatgtatggaagaaaagtgtagttttgaagaagcacgagaagtttttgaaaacactgaaagaacaactgaattttggaagcagtatgttgatggagatcagtgtgagtccaatccatgtttaaatggcggcagttgcaaggatgacattaattcctatgaatgttggtgtccctttggatttgaaggaaagaactgtgaattagatgtaacatgtaacattaagaatggcagatgcgagcagttttgtaaaaatagtgctgataacaaggtggtttgctcctgtactgagggatatcgacttgcagaaaaccagaagtcctgtgaaccagcagtgccatttccatgtggaagagtttctgtttcacaaacttctaagctcacccgtgctgagactgtttttcctgatgtggactatgtaaattctactgaagctgaaaccattttggataacatcactcaaagcacccaatcatttaatgacttcactcgggttgttggtggagaagatgccaaaccaggtcaattcccttggcaggttgttttgaatggtaaagttgatgcattctgtggaggctctatcgttaatgaaaaatggattgtaactgctgcccactgtgttgaaactggtgttaaaattacagttgtcgcaggtgaacataatattgaggagacagaacatacagagcaaaagcgaaatgtgattcgaattattcctcaccacaactacaatgcagctattaataagtacaaccatgacattgcccttctggaactggacgaacccttagtgctaaacagctacgttacacctatttgcattgctgacaaggaatacacgaacatcttcctcaaatttggatctggctatgtaagtggctggggaagagtcttccacaaagggagatcagctttagttcttcagtaccttagagttccacttgttgaccgagccacatgtcttcgatctacaaagttcaccatctataacaacatgttctgtgctggcttccatgaaggaggtagagattcatgtcaaggagatagtgggggaccccatgttactgaagtggaagggaccagtttcttaactggaattattagctggggtgaagagtgtgcaatgaaaggcaaatatggaatatataccaaggtgtcccggtatgtcaactggattaaggaaaaaacaaagctcactgacaaaactcacacatgcccaccgtgcccagctccggaactcctgggcggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgagaattcagacatgataagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttggggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtagcacgtgtcagtcctgctcctcggccacgaagtgcacgcagttgccggccgggtcgcgcagggcgaactcccgcccccacggctgctcgccgatctcggtcatggccggcccggaggcgtcccggaagttcgtggacacgacctccgaccactcggcgtacagctcgtccaggccgcgcacccacacccaggccagggtgttgtccggcaccacctggtcctggaccgcgctgatgaacagggtcacgtcgtcccggaccacaccggcgaagtcgtcctccacgaagtcccgggagaacccgagccggtcggtccagaactcgaccgctccggcgacgtcgcgcgcggtgagcaccggaacggcactggtcaacttggccatggtttagttcctcaccttgtcgtattatactatgccgatatactatgccgatgattaattgtcaacacgtgctgatcagatccgaaaatggatatacaagctcccgggagctttttgcaaaagcctaggcctccaaaaaagcctcctcactacttctggaatagctcagaggcagaggcggcctcggcctctgcataaataaaaaaaattagtcagccatggggcggagaatgggcggaactgggcggagttaggggcgggatgggcggagttaggggcgggactatggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacacctggttgctgactaattgagatgcatgctttgcatacttctgcctgctggggagcctggggactttccacaccctcgtcgagctagcttcgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtgccttgaattacttccacctggctccagtacgtgattcttgatcccgagctggagccaggggcgggccttgcgctttaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaggatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctccagggggctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaggggcctttccgtcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctggagcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaacacgtggtcgcggccgcgccgccaccatggagacagacacactcctgctatgggtactgctgctctgggttccaggttccactggtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaatgactcgagagatctggccggctgggcccgtttcgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtaccggtcatcatcaccatcaccattgagtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaaccagtggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctagaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcccggagacggtcacagcttgtctgtaagcggatgccgggagcagacaagcccgtcagggcgcgtcagcgggtgttggcgggtgtcggggctggcttaactatgcggcatcagagcagattgtactgagagtgcaccatatatgcggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgcca E. Fc DNA sequence (mouse Igκsignal peptide underlined) (SEQ ID NO: 3, which encodes SEQID NO: 4) This is the Fc cassette from pSYN-FIX-030. In addition, there is a separate Fcexpression cassette that was transfected into the cell line in plasmid pSYN-Fc-015 that encodesthe same amino acid sequence, but contains a few noncoding changes. The second copy of Fcencoding sequence enables a better monomer: dimer ratio.

ccaccgtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgcgatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgttggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaac                   cactacacgcagaagagcctctccctgtctccgggtaaa

SEQUENCE TABLE 2 Polypeptide Sequences

A(i). B domain deleted FVIII-Fc chain (19 amino acid signal sequence underlined) (SEQ IDNO: 2)

QSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKA(ii). Fc chain (20 amino acid heterologous signal peptide from mouse Igκchain underlined) (SEQ ID NO: 4)

VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKB. Full length FVIIIFc monomer hybrid (Full length FVIIIFc monomer dimer): created bycoexpressing FVIIIFc and Fc chains. Construct =HC-B-LC-Fc fusion. An Fc expression cassette is cotransfected with full lengthFVIII-Fc to generate the full length FVIIIFe monomer. For the FVIIIFc chain, the Fc sequence isshown in bold; HC sequence is shown in double underline; B domain sequence is shown initalics Signal peptides are underlined.

PENDIEKTDPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHERPQLHHSGDMIFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDLITSSLGPPSMWHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKGKRAHGPALLTEDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFKKVTPLIHDRMLNDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQNSSHLPAASYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELLPKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVESGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNEAIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVEQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWINGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKB(ii). Fc chain (20 amino acid heterologous signal peptide from mouse Igκchain underlined) (SEQ ID NO: 4)

VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK C. FVIII-Fc Heterodimer HybridThis is made by cotransfecting HC-Fc and LC-Fc constructs. Two HC-Fc constructs have beenmade. One has no linker between HC and Fc (HC-Fc) while the other has a 5 amino acid linkerbetween HC and Fc (HC+5-Fc). The FVIII signal peptide was used for the HC-Fc constructs,while the mouse Igκ signal sequence was used for the LC-Fc construct.

NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVFWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

NIAKPRPPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDKTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSME

GPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKC(iii). LC-Fc6His (Fc sequence is shown in bold, signal peptide underlined.) (SEQ ID NO: 12)

WDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKNALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPPCPAPELLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGKD. FIX-Fc chain (SEQ ID NO: 14):(28 amino acid signal sequence underlined, 18 amino acid propeptide double underlined, Fcportion in italics.) The C-terminal lysine is not present in either subunit; this processing is oftenobserved in recombinant proteins produced in mammalian cell culture, as well as with plasmaderived proteins. E. FIXFc-SC subunit: FIX Signal Peptide:-46 MQRVNMIMAESPGLITICLLGYLLSAEC FIX Propeptide: -18 TVFLDHENAN KILNRPKRYNSGKLEEFVQGNLERECMEEKCSFEEAREVFENTERTTEFWKQYVDGDQCESNPCLNGGSCKDDINSYECWCPFGFEGKNCELDVTCNIKNGRCEQFCKNSADNKVVCSCTEGYRLAENQKSCEPAVPFPCGRVSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQSTQSFNDFTRVVGGEDAKPGQFPWQVVLNGKVDAFCGGSIVNEKWIVTAAHCVETGVKITVVAGEHNIEETEHTEQKRNVIRIIPHHNYNAAINKYNHDIALLELDEPLVLNSYVTPICIADKEYTNIFLKFGSGYVSGWGRVFHKGRSALVLQYLRVPLVDRATCLRSTKFTIYNNMFCAGFHEGGRDSCQGDSGGPHVTEVEGTSFLTGIISWGEECAMKGKYGIYTKVSRYVNWIKEKTKLTDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGKMature Fc sequence (corresponding to human IgG1 amino acids 221 to 447, EU numbering)DKTHTCPPCPAPELLGGPSVFLFPPKPICDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

What is claimed is:
 1. A method of treating a bleeding disorder in apatient in need thereof comprising: a) measuring the time betweencontacting of a sample obtained from the patient with an activationmixture dried on a solid substrate and the onset of clotting, therebycalculating the clotting time (Ct), wherein the activation mixturecomprises (i) an activated Factor IX (FIXa) and (ii) a phospholipidmixture, wherein the Ct is used to determine a pharmacokinetic (PK)parameter of a coagulation factor, wherein the coagulation factorcomprises a Factor VIII (FVIII), wherein the PK parameter is a terminalhalf-life (HL) or a time to trough (T), wherein the HL is calculatedaccording to the following formula:HL=−0.693×(T ₂ −T ₁)×A/(Ct₁−Ct₂), wherein A is a constant valuecorresponding to the slope of a Ct versus coagulation factorconcentration dose-response, T₁ and T₂ are times at which Ct ismeasured, and Ct₁ and Ct₂ are Ct values measured at T₁ and T₂,respectively, wherein the T is calculated according to the followingformula:T=−1.44×HL/(A×(Ct_(measured)−Ct_(trough)), wherein A is a constant valuecorresponding to the slope of a Ct versus coagulation factorconcentration dose-response, and HL is the terminal half-life,Ct_(measured) is Ct measured at certain time point, and Ct_(trough) ispatient-specific clot time at trough, and b) administering an effectiveamount of the coagulation factor at a dosing interval based on the PKparameter.
 2. A method of treating a bleeding disorder in a patient inneed thereof comprising: a) measuring the time between contacting of asample obtained from the patient with an activation mixture dried on asolid substrate and the onset of clotting, thereby calculating theclotting time (Ct), wherein the activation mixture comprises (i) anactivated Factor XI (FXIa) and (ii) a phospholipid mixture, wherein theCt is used to determine a pharmacokinetic (PK) parameter of acoagulation factor, wherein the coagulation factor comprises a Factor IX(FIX), wherein the PK parameter is a terminal half-life (HL) or a timeto trough (T), wherein the HL is calculated according to the followingformula:HL=−0.693×(T ₂ −T ₁)×A/(Ct₁−Ct₂), wherein, for each coagulation factor,A is a constant value corresponding to the slope of a Ct versuscoagulation factor concentration dose-response, T₁ and T₂ are times atwhich Ct is measured, and Ct₁ and Ct₂ are Ct values measured at T₁ andT₂, respectively, wherein the T is calculated according to the followingformula:T=−1.44×HL/(A×(Ct_(measured)−Ct_(trough)), wherein for each coagulationfactor, A is a constant value corresponding to the slope of a Ct versuscoagulation factor concentration dose-response, and HL is the terminalhalf-life, Ct_(measured) is Ct measured at certain time point, andCt_(trough) is patient-specific clot time at trough, and b)administering an effective amount of the coagulation factor at a dosinginterval based on the PK parameter.
 3. The method of claim 1, whereinthe correlation between Ct and a coagulation factor level (% Factor) iscalculated based on the following formula:Ct=A×Ln(% Factor)+B wherein, for each coagulation factor, A is aconstant value corresponding to the slope of a Ct versus coagulationfactor concentration dose-response, and B is a patient-specific off-setvalue.
 4. The method of claim 1, further comprising monitoring theefficacy of the coagulation factor administered to the patient.
 5. Themethod of claim 1, wherein the PK parameter is terminal half-life (HL).6. The method of claim 1, wherein the PK parameter is time to trough(T).
 7. The method of claim 6, wherein the patient is administered a newdose of the coagulation factor every T interval.
 8. The method of claim1, wherein the sample is selected from the group consisting of wholeblood, citrated or equivalently stabilized blood, plasma, and otherfluid sample containing or suspected of containing a coagulation factor.9. The method of claim 8, wherein the sample further comprises an addedinhibitor.
 10. The method of claim 1, wherein the Ct correlates with atherapeutically efficacious treatment.
 11. The method of claim 1,wherein the FVIII is a chimeric FVIII polypeptide comprising FVIII and anon-FVIII polypeptide, wherein the non-FVIII polypeptide increases ahalf-life of the FVIII and comprises Fc, albumin, a PAS sequence,transferrin, C-terminal peptide of hCG (CTP), polyethylene glycol (PEG),hydryoxyethyl starch (HES), or two or more combinations thereof.
 12. Themethod of claim 2, wherein the FIX is a chimeric FIX polypeptidecomprising FIX and a non-FIX polypeptide, wherein the non-FIXpolypeptide increases a half-life of the FIX and comprises Fc, albumin,a PAS sequence, transferrin, C-terminal peptide of hCG (CTP),polyethylene glycol (PEG), hydryoxyethyl starch (HES), or two or morecombination thereof.
 13. The method of claim 11, wherein the chimericFVIII polypeptide comprises a monomer dimer hybrid comprising the FVIIIand an Fc.
 14. The method of claim 12, wherein the chimeric FIXpolypeptide comprises a monomer dimer hybrid comprising the FIX and anFc.
 15. The method of claim 2, wherein the correlation between Ct and acoagulation factor level (% Factor) is calculated based on the followingformula:Ct=A×Ln(% Factor)+B, wherein A is a constant value corresponding to theslope of a Ct versus coagulation factor concentration dose-response, andB is a patient-specific off-set value.
 16. The method of claim 2,further comprising monitoring the efficacy of the coagulation factoradministered to the patient.
 17. The method of claim 2, wherein the PKparameter is terminal half-life (HL).
 18. The method of claim 2, whereinthe PK parameter is time to trough (T).
 19. The method of claim 18,wherein the patient is administered a new dose of the coagulation factorevery T interval.
 20. The method of claim 2, wherein the sample isselected from the group consisting of whole blood, citrated orequivalently stabilized blood, plasma, and other fluid sample containingor suspected of containing a coagulation factor.
 21. The method of claim20, wherein the sample further comprises an added inhibitor.
 22. Themethod of claim 2, wherein the Ct correlates with a therapeuticallyefficacious treatment.